Compositions, methods and kits for detection of an antigen on a cell and in a biological mixture

ABSTRACT

The present invention relates to novel methods for detecting at least one member of a known binding pair in a sample, including a cell, where one member of the pair (termed the “receptor”) is expressed by a bacteriophage, which phage is then used to detect the presence of the other member of the pair (termed the “ligand” or “target”). Rather than detecting the binding of at least one phage using antibody-based technology, the present invention relates to detecting the nucleic acid associated with the phages. In one aspect, the invention relates to identifying at least one antigen-bearing moiety (e.g., a red blood cell antigen) of interest present on a cell, e.g., a red blood cell, using antibody-displaying bacteriophages, using antiglobulin reagent-displaying bacteriophages and detecting at least one nucleic acid associated with the phage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of International ApplicationNo. PCT/US03/29231, filed on Sep. 18, 2003, now published asWO2004/027028, which claims priority of U.S. Provisional PatentApplication 60/411,693, filed Sep. 18, 2002, both of which applicationsare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Each year in the United States alone, hundreds of millions of red bloodcell (RBC) antigen typings are performed on donated units of blood andthe patients that are to receive them. In addition, equivalent numbersof patient antisera are screened for the presence of pre-existinganti-RBC antibodies, the specificities of which must be identified priorto the selection of compatible blood. The technology used in blood banksfor doing these tests is essentially the same as the one demonstrated byLandsteiner over 100 years ago—the agglutination of RBCs by anappropriate antisera. Assay systems of this type are labor intensive andtypically require teams of highly-trained medical technologists manuallyshaking test tubes over magnifying mirrors and assessing agglutinationpatterns by eye. Consequently, blood banks require significantly morebench technologists per test than any other type of clinical laboratory,as reflected in the 10- to 100-fold greater cost per test for thetransfusion laboratory than those for other areas of laboratorymedicine. In addition, blood donation facilities, blood banks, andhospital transfusion services across the country are facing a growingshortage of skilled staff to perform such tests due to the lack ofqualified and interested candidates. This is particularly concerninggiven the extraordinary importance of accurate pre-transfusion testingand the ability to provide blood components to patients in a timely,often emergent, basis.

As opposed to other forms of laboratory testing such as those inclinical chemistry, coagulation, and hematology, blood bank testing hasdefied the development of rapid, high-throughput automation. The methodsfor blood bank automation that are currently available require, inessence, the use of a machine that detects the agglutination of redcells, but agglutination (or some variant thereof) is still theend-point much as it was nearly 100 years ago. Reasons for thedifficulty in developing truly automated blood typing systems aremultiple, but in large part have to do with the need to work with intactcells in order to detect the presence of specific polymorphic moleculeson their surfaces. This is in contrast to other laboratory tests thatsimply count numbers of cells or measure the concentrations of solubleplasma proteins or electrolytes.

While it is true that flow cytometric testing also detects cell-surfacephenotype, the indications for such tests do not, in general, requirerapid real-time results such as those required in transfusion medicinewhere the goal is to prevent the transfusion of incompatible blood,often during emergencies such as trauma or unanticipated surgery, wheretime and accuracy are of the essence. Furthermore, essential differencesin the nature of blood bank testing have precluded the development of“point-of-care” testing devices, such as those now available for glucoseor electrolyte determinations or for the rapid “on-the-scene” diagnosisof myocardial infarction. The development of novel blood bank testingmethods could lead to the development of small, portable devices forpre-transfusion testing that could facilitate “point-of-care” (e.g.,battlefield) testing not possible using conventional approaches.

Another significant issue in blood banking testing is the growingunavailability of complete panels of high-quality immunological reagentsfor typing. Supplies of conventional sources come from donated humanpolyclonal antisera that are difficult to quality control and aredwindling in supply due to growing ethical concerns regarding thedeliberate hyperimmunization of reagent donors. Because immune responsesto many blood group antigens are mounted only in humans (who lack theparticular antigen) and not in animals (e.g., mice, whose immune systemsgenerally cannot detect the subtle human polymorphisms to which theantisera needs to be directed), efforts to produce monoclonal typingreagents have required the ability to transform human B-cells, which isa very inefficient and expensive endeavor. Therefore, the availabilityof endless supplies of well-characterized monoclonal RBC antibodies,analogous to those which revolutionized the automation of otherimmunological-based assays, such as those for endocrinology orinfectious diseases, has been problematic in the field of transfusionmedicine.

More than 20 million units of blood are collected in the United Statesannually, with worldwide collections exceeding 40 million units. Bloodcollection centers (e.g., American Red Cross, hospital-based donorcenters), hospitals, and other blood banks and transfusion centers allhave on-going needs to type blood quickly and accurately in ahigh-throughput manner. Small, automated, blood typing instruments wouldalso have “point-of-care” applications in physician offices such asthose of obstetricians in which a patient's Rh type needs to bedetermined in order to properly administer Rh(D)-immune globulin. Eachunit of blood that is collected is typed for at least 3 (i.e., A, B,Rh(D)) antigens and often the blood is tested for detection of many moreantigens (e.g., Rh(C), Rh(c), Rh(E), Rh(e), K, Fy^(a), Fy^(b), M, N, S,s, Jk^(a), Jk^(b), and the like).

Upon receipt of units by a blood bank, standards require that each unitbe retested for A and B to ensure proper labeling. Each collected unitof blood is separated into red cells, platelets, and plasma in order totreat 3 different patients with different needs. Approximately twice asmany patients are typed for A, B, and Rh(D) (and often other antigens)than those who actually receive blood (i.e., crossmatch/transfusionratio is approximately 2). In addition, blood samples are collectedevery seventy-two hours on hospitalized patients in order to have freshsamples available for cross-matching purposes such that many patientsare typed and retyped many times during their hospitalization.Therefore, the number of blood typings performed worldwide annually isin the hundreds of millions of tests.

As noted previously, essentially all methods for RBC typing, whethermanual or automated, use agglutination as the endpoint. Thedisadvantages of manual methods include labor costs, low throughput, andhuman error. Disadvantages of current automated methods includeinability to multiplex testing reactions and relatively low throughputwhen compared to other laboratory testing. Additionally, significantdisadvantages of both current manual and automated methods include theirreliance on conventional sources of antisera, which sources aredwindling in supply and can potentially transmit human disease, or thefew human or mouse hybridoma-produced antibodies which are difficult andexpensive to produce. The present invention provides endless supplies ofinexpensive phage-displayed anti-RBC reagents that can be used not onlyin an automated “phenotyping-by-reagent genotyping” technology asdisclosed herein, but that are also compatible with conventional manualand automated agglutination methods using anti-M13 antibody as theagglutinating (i.e. “Coombs”) agent (e.g., U.S. Pat. No. 5,985,543, toSiegel).

In sum, there is a long-felt and acute need for improved blood typingmethods and reagents therefore, which will allow the automation of suchtests thereby lowering costs, improving efficiency and accuracy, andobviating the need for current difficult to obtain reagents. The presentinvention meets these needs.

BRIEF SUMMARY OF THE INVENTION

The invention includes a method of detecting the presence of anantigen-bearing moiety on a cell. The method comprises, a) contacting acell with a bacteriophage expressing an antibody known to specificallybind with the antigen-bearing moiety wherein the bacteriophage comprisesa nucleic acid and wherein the sequence of the nucleic acid is at leastpartially known; b) denaturing any bacteriophage specifically bound withthe cell to release the nucleic acid; and c) detecting the nucleic acidusing a melting curve profile, wherein detecting the nucleic aciddetects the presence of the antigen-bearing moiety on the cell, therebydetecting the presence of the antigen-bearing moiety on the cell.

In another aspect, the melting curve profile is generated using acapillary PCR fluorescent device.

In yet another aspect the capillary PCR fluorescent device includes acomponent for polymerase chain reaction and a component forspetrophotometric detection.

In one aspect, the method further comprises washing the cell betweenstep (a) and step (b).

In yet another aspect, the cell is a red blood cell and theantigen-bearing moiety is a red blood cell antigen.

In a further aspect, the red blood cell antigen is selected from thegroup consisting of A, B, Rh(D), Rh(C), Rh(c), Rh(E), Rh(e), K, Fy^(a),Fy^(b), M, N, S, s, Jk^(a), and Jk^(b).

In one aspect, the cell is a white blood cell and the antigen-bearingmoiety is selected from the group consisting of a lymphocyte antigen, amonocyte antigen, and a granulocyte antigen.

The invention includes a method of detecting the presence of at leasttwo different antigen-bearing moieties on a cell. The method comprises:a) contacting a cell with a first bacteriophage expressing an antibodyknown to specifically bind with a first antigen-bearing moiety whereinthe first bacteriophage comprises a first nucleic acid and wherein thesequence of the first nucleic acid is at least partially known; b)contacting the cell with a second bacteriophage expressing an antibodyknown to specifically bind with a second antigen-bearing moiety whereinthe second bacteriophage comprises a second nucleic acid and wherein thesequence of second the nucleic acid is at least partially known andwherein the sequence of the first nucleic acid is detectably differentfrom the sequence of the second nucleic acid; c) detecting the bindingof the first bacteriophage with the antigen-bearing moiety by detectingthe presence of the first nucleic acid using a melting curve profile,wherein detecting the first nucleic acid detects the presence of thefirst antigen-bearing moiety on the cell; d) detecting the binding ofthe second bacteriophage with the antigen-bearing moiety by detectingthe presence of the second nucleic acid using a melting curve profile,wherein detecting the second nucleic acid detects the presence of thesecond antigen-bearing moiety on the cell; thereby detecting thepresence of at least two different antigen-bearing moieties on the cell.

In one aspect, the method further comprises washing the cell betweenstep (a) and step (b).

In another aspect, the melting curve profile is generated using acapillary PCR fluorescent device.

In yet another aspect the capillary PCR fluorescent device includes acomponent for polymerase chain reaction and a component forspetrophotometric detection.

In yet another aspect, the cell is a red blood cell and theantigen-bearing moiety is a red blood cell antigen.

In a further aspect, the red blood cell antigen is selected from thegroup consisting of A, B, Rh(D), Rh(C), Rh(c), Rh(E), Rh(e), K, Fy^(a),Fy^(b), M, N, S, s, Jk^(a), and Jk^(b).

In one aspect, the cell is a white blood cell and the antigen-bearingmoiety is selected from the group consisting of a lymphocyte antigen, amonocyte antigen, and a granulocyte antigen.

In yet another aspect, the cell is a platelet and the antigen-bearingmoiety is a platelet antigen.

In a further aspect, the platelet antigen is selected from the groupconsisting of HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a,HPA-4b, HPA-5a, HPA-5b, HPA-6b, HPA-7b, HPA-8b, HPA-9b, HPA-10b,Gov^(a), and Gov^(b).

The invention includes a kit for detecting the presence of anantigen-bearing moiety on a cell. The kit comprises a bacteriophageexpressing an antibody known to specifically bind with theantigen-bearing moiety wherein the bacteriophage comprises a nucleicacid and wherein the sequence of the nucleic acid is at least partiallyknown. The kit further comprises an applicator, and an instructionalmaterial for the use of the kit.

In one aspect, the antigen-bearing moiety is a red blood cell antigenselected from the group consisting of A, B, Rh(D), Rh(C), Rh(c), Rh(E),Rh(e), K, Fy^(a), Fy^(b), M, N, S, s, Jk^(a), and Jk^(b).

In a further aspect, a kit further comprises a PCR primer.

In yet a further aspect, the sequence of the primer is selected from thegroup consisting of the sequence of SEQ ID NO:1 and the sequence of SEQID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a diagrammatical outline of technical plan illustrating use of(A) phage-displayed anti-RBC antibodies, (B) phage DNA amplification,and (C) phage DNA detection.

FIG. 2 is a diagram of a schematic representation of anti-B (top) andanti-Rh(D) (bottom) phage-displayed human monoclonal RBC antibodies.

FIG. 3 is an image depicting phenotyping RBCs for the blood group B andRh(D) antibodies in a multiplex phage antibody assay. Four possible RBCphenotypes (positive or negative for the blood group B antigen andpositive or negative for the Rh(D) antigen) were incubated with phagedisplayed anti-B alone, anti-Rh(D) alone, anti-B and anti-Rh(D)together, or buffer. After washing away unbound phage reagent, RBCs wereresuspended in anti-M13 phage antibody, an aliquot of the cellsuspension was removed, diluted 200-fold in water, and 2-microliters ofthe diluted phage/lysed RBCs were subjected to PCR. The balance of theanti-M13 resuspended RBC samples were placed in microtiter plate wellsand assayed for agglutination as described elsewhere herein (e.g.,Siegel et al., 1997, J. Immunol. Meth. 206:73-85). Note thatagglutination (top panel, wells with large crosslinked cell pellets)only occurs with the appropriate antibody/cell phenotype combination asexpected. Most notably, only the appropriate antibody sequence wasdetected (1600-bp product with RBCs that expressed blood group Bantigen; 1000-bp product with RBCs that expressed the Rh(D) antigen) andthere was no detectable background (i.e., no anti-B DNA product withtype O RBCs which do not express group A or B antigens; and noanti-Rh(D) DNA product was detected using Rh(D)-negative cells). For PCRamplification of the inserts, the forward primer (“5-prime LC”) was asfollows: 5′-AAGACAGCTATCGCGATTG-3′ (SEQ ID NO:1); and the reverse primer(“GBACK”) was as follows: 5′-GCCCCCTTATTAGCGTTTGCCATC-3′ (SEQ ID NO:2).

FIG. 4, comprising FIGS. 4A and 4B, depict a diagram illustratingvarious phagemid constructs for anti-B-expressing phage particles (FIG.4A) and anti-Rh(D)-expressing phage particles (FIG. 4B). The diagramillustrates cloning of inserts of about 140 basepairs in size (morespecifically, 142 bp) into the anti-B phagemid (“B140”) or anti-Rh(D)phagemid (“D140”) downstream of the 20-bp T7 RNA polymerase promotersite. The 142-bp inserts are identical except for an internal 33-bpregion to which B-or Rh(D)-specific molecular beacons or microarrayedoligos hybridize (“B-Beacon/Oligo” and “D-Beacon/Oligo”, respectively).B140 and D140 can be amplified by PCR with an identical set ofoligonucleotide primers (“PCR-F” and “PCR-R”) or transcribed using T7RNA polymerase. The sequence of the “B140” insert is5′-TGCTATGTCACTTCCCCTTGGTTCTCTCATCTGGCCTGGTGCAATAGGCCCTGCATGCACTGGATGCACTCTATCCCATTCTGCAGCTTCCTCATTGATGGTCTCTTTTAACATTTGCATGGCTGCTTGATGTCCCCCCACT-3′ (SEQ ID NO:3) and the sequence ofthe “D140” insert is5′-TGCTATGTCACTTCCCCTTGGTTCTCTCATCTGGCCTGGTGCAATAGGCCCTGCATGCACTGGATGCACTCTGTTTTACCTCATTATCCTTCTGCCAGCGCTAGCTTTTAACATTTGCATGGCTGCTTGATGTCCCCCCACT-3′ (SEQ ID NO:4). The forward PCRprimer (“PCR-F”) is: 5′-TGCTATGTCACTTCCCCTTGGTTCTCT-3′ (SEQ ID NO:5) andthe reverse PCR primer (“PCR-R”) sequence is:5-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3′ (SEQ ID NO:6). The B-Beacon andD-Beacon sequences are as follows, showing the fluorescent derivativesand the stem structures in lower case. The “B-Beacon” sequence is asfollows: 6-FAM-gcgagcATCCCATTCTGCAGCTTCCTCATTGATGGTCTCgctcgc-DABCYL (SEQID NO:7. The “D-Beacon” is:TAMRA-cgagcGTTTTACCTCATTATCCTTCTGCCAGCGCTAGCgctcgc-DABCYL (SEQ ID NO:8).The upper case letters in the beacon sequences represent the respectivesequences in B140 and D140 to which the beacons anneal. Therefore, theupper case letters are the sequences of the oligonucleotides that areused for the DNA array detection. That is, a B-oligo is:5′-ATCCCATTCTGCAGCTTCCTCATTGATGGTCTC-3′ (SEQ ID NO:9), and a “D-oligo”is: 5′-GTTTTACCTCATTATCCTTCTGCCAGCGCTAGC-3′ (SEQ ID NO:10).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By the term “antigen-bearing moiety” as used herein, is meant a moleculeto which an antibody binds. The antigen-bearing moiety may be a membranebound protein which is selected from the group consisting of an antigenand a receptor. In another aspect, the membrane bound protein is anantigen, such as a red blood cell antigen, such as Rh antigen. When theantigen-bearing moiety is a carbohydrate, it may be a carbohydrateexpressed on a glycolipid, for example, a P blood group antigen or otherantigen.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding a protein,or to a sequence which is substantially homologous to the non-codingstrand. As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

The terms “bacteriophage” and “phage” are used interchangeably hereinand refer to viruses which infect bacteria. By the use of the terms“bacteriophage library” or “phage library” as used herein, is meant apopulation of bacterial viruses comprising heterologous DNA, i.e., DNAwhich is not naturally encoded by the bacterial virus.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for administering the bacteriophage expressing a receptor(e.g., an antiglobulin reagent, an antibody, an anti-antibody, and thelike), a cell, a sample, primers, molecular beacon probe, dNTPs, T7 RNApolymerase, and the like, of the invention to a cell, a sample, and thelike.

“Biological sample,” or simply “sample”, as that term is used herein,means a sample, such as one that is, but need not be, obtained from ananimal, which sample is to be assessed for the presence of a biologicalorganism, or component thereof, such that the sample can be used toassess the presence, absence and/or level, of an antigen, or ligand, ofinterest according to the methods of the invention. Such sampleincludes, but is not limited to, any biological fluid (e.g., blood,lymph, semen, sputum, saliva, phlegm, tears, and the like), fecalmatter, a hair sample, a nail sample, a brain sample, a kidney sample,an intestinal tissue sample, a tongue tissue sample, a heart tissuesample, a mammary gland tissue sample, a lung tissue sample, an adiposetissue sample, a muscle tissue sample, and any sample obtained from ananimal that can be assayed for the presence or absence of an antigen.Further, the sample can comprise an aqueous sample (e.g., a watersample) however obtained, to be assessed for the presence of anorganism, or a component thereof, such as a drinking water sample,before or after any treatment, wherein the presence of a biologicalorganism (e.g., a Cryptosporidium organism) is assessed.

As used herein, the term “fragment” as applied to a nucleic acid, mayordinarily be at least about 20 nucleotides in length, preferably, atleast about 30 nucleotides, more typically, from about 40 to about 50nucleotides, preferably, at least about 50 to about 80 nucleotides, evenmore preferably, at least about 80 nucleotides to about 90 nucleotides,yet even more preferably, at least about 90 to about 100, even morepreferably, at least about 100 nucleotides to about 150 nucleotides, yeteven more preferably, at least about 150 to about 200, even morepreferably, at least about 200 nucleotides to about 250 nucleotides, yeteven more preferably, at least about 250 to about 300, more preferably,from about 300 to about 350 nucleotides, preferably, at least about 350to about 360 nucleotides, and most preferably, the nucleic acid fragmentwill be greater than about 365 nucleotides in length.

As used herein, the term “fragment” as applied to a polypeptide, mayordinarily be at least about 20 amino acids in length, preferably, atleast about 30 amino acids, more typically, from about 40 to about 50amino acids, preferably, at least about 50 to about 80 amino acids, evenmore preferably, at least about 80 amino acids to about 90 amino acids,yet even more preferably, at least about 90 to about 100, even morepreferably, at least about 100 amino acids to about 120 amino acids, andmost preferably, the amino acid fragment will be greater than about 123amino acids in length.

By the term “Fab/phage” as used herein, is meant a phage particle whichexpresses the Fab portion of an antibody.

By the term “scFv/phage” are used herein, is meant a phage particlewhich expresses the Fv portion of an antibody as a single chain.

“Phage,” or “phage particle,” as these terms are used herein, includethat contain phage nucleic acid encoding, inter alia, an antibody. Thisis because, as would be appreciated by the skilled artisan, unlikepeptide phage display (where the peptide DNA insert is small and it isactually cloned into the phage DNA), the larger scFv or Fab DNA insertsare actually cloned into, among other things, a plasmid. Thus, thenucleic acid encoding the antibody, e.g., a plasmid such as, but notlimited to, pComb3, not only comprises a plasmid origin of replication,but also a phage (e.g., M13) origin of replication sequence and an M13packaging sequence, so that when the nucleic acid is produced, a helperphage can be used to provide the required phage (e.g., M13) proteins intrans to make “phage-like” particles. That is, these particles resemblephage on the outside, but on the inside they contain plasmid (alsoreferred to as a “phagemid”) DNA. In other words, the phagemid DNA neednot encode any M13 phage proteins, except a piece of M13 gene III fusedto the DNA for antibody or peptide. Thus, it should be understood thatthe terms “phage,” “phage particle,” “phage-like particle” and“phagemid” are used interchangeably herein.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated,then the animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 5′ATTGCC3′ and 5′TATGGC3′ share 50%homology.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the nucleic acid,peptide, and/or compound of the invention in the kit for detecting thepresence of an antigen-bearing moiety on a cell of interest, and/or fordetecting an autoantibody in serum. The instructional material of thekit may, for example, be affixed to a container that contains thenucleic acid, peptide, and/or compound of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or compound. Alternatively, the instructional material may beshipped separately from the container with the intention that therecipient uses the instructional material and the compoundcooperatively.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids that have beensubstantially purified from other components that naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA that is part of a hybrid gene encoding additionalpolypeptide sequence.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred toas a “recombinant host cell.” A gene that is expressed in a recombinanthost cell wherein the gene comprises a recombinant polynucleotide,produces a “recombinant polypeptide.”

A “recombinant polypeptide” is one that is produced upon expression of arecombinant polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell.

Numerous vectors are known in the art including, but not limited to,linear polynucleotides, polynucleotides associated with ionic oramphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term shouldalso be construed to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example,polylysine compounds, liposomes, and the like. Examples of viral vectorsinclude, but are not limited to, adenoviral vectors, adeno-associatedvirus vectors, retroviral vectors, and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

Preferably, when the nucleic acid encoding the desired protein furthercomprises a promoter/regulatory sequence, the promoter/regulatory ispositioned at the 5′ end of the desired protein coding sequence suchthat it drives expression of the desired protein in a cell. Together,the nucleic acid encoding the desired protein and itspromoter/regulatory sequence comprise a “transgene.”

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “polyadenylation sequence” is a polynucleotide sequence which directsthe addition of a poly A tail onto a transcribed messenger RNA sequence.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

A “portion” of a polynucleotide means at least at least about twentysequential nucleotide residues of the polynucleotide. It is understoodthat a portion of a polynucleotide may include every nucleotide residueof the polynucleotide.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe” refers to a polynucleotide that is capable of specificallyhybridizing to a designated sequence of another polynucleotide. A probespecifically hybridizes to a target complementary polynucleotide, butneed not reflect the exact complementary sequence of the template. Insuch a case, specific hybridization of the probe to the target dependson the stringency of the hybridization conditions. Probes can be labeledwith, e.g., chromogenic, radioactive, or fluorescent moieties and usedas detectable moieties.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, the term “reporter gene” means a gene, the expression ofwhich can be detected using a known method. By way of example, theEscherichia coli lacZ gene may be used as a reporter gene in a mediumbecause expression of the lacZ gene can be detected using known methodsby adding a chromogenic substrate, e.g.,o-nitrophenyl-β-D-galactopyranoside, to the medium (Gerhardt et al.,eds., 1994, Methods for General and Molecular Bacteriology, AmericanSociety for Microbiology, Washington, D.C., p. 574).

A “receptor” is a compound that specifically binds with a ligand. Thisterm includes a protein, such as an antibody, an antiglobulin reagent,and the like, that when expressed by a phage and contacted with itscognate ligand, binds specifically therewith.

The term “ligand,” as used herein, refers to any protein or proteinsthat can interact with a receptor binding domain, thus having a “bindingaffinity” for such domain. Ligands can be soluble or membrane bound, andthey can be a naturally occurring protein, or synthetically orrecombinantly produced. The “ligand” can also be a nonprotein moleculethat acts as ligand when it interacts with the receptor binding domain.Interactions between the ligand and receptor binding domain include, butare not limited to, any covalent or non-covalent interactions. Thereceptor binding domain is any region of the receptor molecule thatinteracts directly or indirectly with the ligand.

By the term “specifically binds,” as used herein, is meant a molecule,e.g., a protein, a nucleic acid, an antibody, a compound, and the like,which recognizes and binds a specific molecule, but does notsubstantially recognize or bind other molecules in a sample. Forinstance, an antibody which recognizes and binds a cognate ligand (i.e.,an antigen-bearing moiety present on a cell) in a sample, but does notsubstantially recognize or bind other molecules in the sample.

To “treat” a disease as the term is used herein, means to reduce thefrequency of the disease or disorder reducing the frequency with which asymptom of the one or more symptoms disease or disorder is experiencedby an animal.

Description

The invention relates to methods for detecting the presence of amolecule of interest on a cell or in a biological sample. Typically, ared blood antigen expressed on a RBC surface is detected, but theinvention encompasses detecting the presence of numerous antigens ofinterest on a wide plethora of cells, including, but not limited to, redand white blood cells, as well as platelets, and cells used fortransplantation therapy, and the identification of antigens on cells forforensic purposes (e.g., hair, skin, nail, sperm, saliva, and othercells), among many other uses.

The invention also relates to detection of an antigen of interest in abiological sample. Such a sample includes an aqueous sample to detectthe presence of any organism, or component thereof, in the sample.

The invention relates to using an antibody, specific for a knownantigen, displayed by a phage (e.g., an M13, T7, lambda, eukaryotic, andthe like), to detect the presence of the antigen on a cell or in abiological sample. More specifically, phage specifically bound with acell are detected by assaying for the nucleic acid contained in thephage particle. That is, the nucleic acid sequence of the nucleic acidcontained in the phagemid is at least partially known, such thattechniques for detecting nucleic acids can be used to assess thepresence of the sequence, thereby detecting, in a novel process referredto herein as “phenotyping-by-reagent-genotyping”, the antigen.

Essentially, the bacteriophage nucleic acid acts like a tag fordetecting an antigen recognized by the antibody encoded by the phage. Inthis way, the high sensitivity and high throughput screening propertiesof nucleic acid detection methods can be applied to the immunologicaldetection of an antigen, thereby combining the advantages of bothtechnologies. The crucial features of this approach are that thespecificity of the antibody displayed by the bacteriophage and thenucleic acid sequence, or a portion thereof, of the DNA contained withinthe phage, both be known. It would be understood, based upon thedisclosure provided herein, that the precise nature of the antigen, beit a protein, carbohydrate, lipid, or any other compound, recognized bythe antibody, need not be known, only that the specificity of theantibody for that antigen be known. For instance, where an antibody isknown to bind with and identify a cancer cell (or any cell associatedwith a disease), but not bind with an otherwise identical cell that isnot cancerous (or associated with a disease), the antibody can be usedto detect a cancer (or disease state) using the methods of theinvention. That is, the antibody binding with a test cell or abiological sample, can be detected by detecting the nucleic acid presentin the phage particle encoding the antibody portion, thereby detecting acancer cell, without having to know the precise nature of the antigenpresent on the cancer (or disease-associated) cell.

The invention further relates to detection of multiple antigens ofinterest on a cell in a single tube assay. That is, bacteriophage thatencode antibodies specific for at least two different antigens can beused to detect those antigens on a cell. More specifically, each phageencodes an antibody that specifically binds with a known antigen andeach phage encodes an antibody that recognizes a different antigen, orantigen-moiety. Further, each phage contains a DNA molecule comprising asequence that is known, wherein the sequence differs between the phage.Using this approach, the presence of a plurality of antigens of interestcan be readily assessed by simply using a panel of phage, eachdisplaying an antibody specific for one of the antigens, where thenucleic acid molecule of each phage comprises a known sequence that isdistinguishable from that of any other phage in the panel. In this way,multiple antigens can be assayed for using a single reaction step. This“multiplexing” method is not possible using conventional methods thatidentify the binding of antigen-specific antibodies to a cell since thesecondary anti-antibody antibody used to detect the antigen-specificantibodies typically cross-reacts with all the antigen-bindingantibodies, or it cannot be determined which antigen-specific antibodythe second antibody is bound with. In the case of conventional methodsfor phenotyping red blood cells, in which antibodies directlyagglutinate the appropriate cell type (i.e., no secondary antibodyneeded), if mixed together, it would likewise not be possible todetermine which antigen-specific antibody was responsible for theagglutination. This multiplex approach allows the rapid simultaneousdetection of a plurality of antigens using only a single sample.

Further, the invention relates to identification of anti-red blood cellantibodies in serum. That is, a panel of RBCs, expressing various knownantigens on their surfaces, can be contacted with a serum sample.Reagent RBCs, expressing characterized antigens, are commerciallyavailable (e.g., Johnson & Johnson, Raritan, N.J.). The cells are thenwashed to remove any antibodies non-specifically adhering to the cellsand the cells are then contacted with bacteriophage displaying ananti-globulin reagent.

Additionally, autoantibodies present in a patient can be detected byobtaining RBCs from the patient, washing them to remove any antibodiesand/or complement that is non-specifically bound with the cells, and thecells can then be contacted with a phage expressing an antihumanglobulinreagent. Thus, by detecting a nucleic acid sequence contained by thephage, the presence of autoantibody on the patient cells, as well as thepresence of complement deposited on the cells due to the autoantibody,can be readily detected according to the novel“phenotyping-by-reagent-genotyping” methods disclosed herein.

Conventionally, screening and identification of serum antibodies usingreagent red cells displaying known antigens is referred to in the art asan “antiglobulin test”, one such test is a Coombs reaction. These assaysdetect the presence of an antibody, or complement deposited thereby, ona cell of interest. Because complement, while not an antibody, isconsidered a “globulin”, the reagents used to detect antibodies and/orcomplement are referred to in the art as “antiglobulin” reagents.

These assays, which detect antibodies and/or complement fragments (e.g.,C3d) on patient red cells to detect anti-red cell autoantibodies, or thecomplement they deposit, and also to detect patient alloantibodies, orthe complement they deposit, can be used to identify autoantibodies,alloantibodies, or both, that could be destroying autologous cells ortransfused cells in a hemolytic transfusion reaction.

As used herein, an “antiglobulin reagent” is a reagent that can detectantibodies, complement, or both. Thus, the present invention includes,as would be understood by one skilled in the art armed with theteachings provided herein, antiglobulin reagents comprising, amongothers, e.g., anti-antibody antibodies, anti-complement antibodies,Protein A, Protein G, or Protein L, that is, the invention encompassesexpression by phage of a wide plethora of reagents that would beunderstood by the skilled artisan to specifically bind with a globulin,such as antibody, complement, and the like. That is, the presentinvention includes using an antiglobulin reagent expressed by a phageincluding, but not limited to, an “anti-antibody antibody”, ananti-complement, and any reagent known to bind a globulin (e.g., anantibody, complement, and the like). Additionally, phage expressingProtein A, or an immunoglobulin-binding domain thereof, have beendescribed previously (e.g., Djojonegoro et al., 1994, Bio/Technol.12:169-172). Such antiglobulin reagent-expressing phage can be used inthe methods disclosed herein as would be understood by one skilled inthe art armed with the teachings provided herein.

The invention relates to identifying autoantibodies in a serum sampleobtained from a patient, or autoantibodies or complement fragmentspre-deposited on patient cells in vivo, both characteristics of adisease such as, but not limited to, autoimmune hemolytic anemia. Thatis, serum obtained from the patient is contacted with an aliquot ofreagent RBCs, such as those that are commercially available. RBCautoantibodies bind to common antigens present on essentially all redcells, not just of the patient. Thus, the patient cannot be transfusedwith blood from another human since the autoantibodies present in thepatient serum with also react with the donor RBCs. Because the patient'sRBCs are already be coated with the autoantibodies, those autoantibodiesalready on the cells from having been bound in vivo can be detectedaccording to the methods of the invention by assaying the cells directlyusing antihumanglobulin reagent expressed on a phage. Alternatively,detecting autoantibodies is performed the same way as is detection ofalloantibodies—by contacting the patient serum with reagent red cells.In the case of alloantibodies, only certain reagent RBCs will bind theantibodies, and knowing the precise phenotype of those cells identifiesthe antigen specificity. In the case of autoantibodies, typically allreagent red cells will bind the antibodies because the autoantigens arepresent on all cells. Any antibody specifically bound with the RBCs isthen detected according to the methods of the invention such as, as morefully disclosed elsewhere herein, by contacting the cells with a phageexpressing an antiglobulin reagent and detecting the binding of thephage with the cells by detecting a nucleic acid contained by the phage,i.e., by performing “phenotyping-by-reagent-genotyping” according to themethods of the invention. In this way, autoantibodies present in humanserum can be readily detected using the methods disclosed hereinanalogous to the conventional “indirect antiglobulin test”. Furthermore,by contacting patient RBCs with antiglobulin-expressing phage particlesand detecting the binding of the phage with the cells by detecting anucleic acid contained by the phage, one can detect the presence of invivo-deposited autologous antibodies and/or complement fragments onpatient RBCs. This assay is analogous to the conventional “directantiglobulin test”.

Further, the invention relates to performing compatibility testingbetween patient serum and red cells drawn from prospective units ofblood to be transfused to the patient (i.e., patient/donor“crossmatching”). That is, an aliquot of RBCs from a prospective unit ofdonor blood can be contacted with a serum sample from a potentialtransfusion recipient. The cells are then washed to remove anyantibodies non-specifically adhering to the cells and the cells are thencontacted with bacteriophage displaying an antiglobulin reagent. Thus,the present invention provides methods for detecting an alloantibody ina patient that is to be transfused thereby allowing proper patient/donorcrossmatching to prevent incompatible transfusion.

I. Methods

A. Methods of Detecting an Antigen

The invention includes a method for detecting the presence of anantigen-bearing moiety on a cell. The method comprises contacting a cellwith a bacteriophage expressing an antibody that is known tospecifically bind with the antigen-bearing moiety when it is present ona cell. Such phage-displayed antibodies, as well as methods for theirproduction, are well-known in the art, and are described in, amongothers, U.S. Pat. Nos. 5,876,925, 5,985,543, and 6,255,455, all toSiegel. These antibody-displaying bacteriophage are exemplified hereinby phage displaying anti-Rh(D) and anti-B specific antibodies. However,the skilled artisan would understand, based upon the disclosure providedherein, that the invention is not limited to these, or any other,particular antibodies displayed on the specific bacteriophage disclosedherein. Rather, the antibody displayed by the phage can be specific forany cell component and techniques for producing phage-displayingantibodies to antigens of interest are well-known in the art, and areencompassed in the present invention.

The procedures for making a bacteriophage library comprisingheterologous DNA are well known in the art and are described herein in,as well as in for example, in Sambrook et al., supra. Bacteriophagewhich encode a desired antibody can be engineered such that the antibodyprotein is displayed on the surface thereof in such a manner that it isavailable for binding to its corresponding binding protein, e.g., theantigen against which the antibody is directed. Thus, when bacteriophagewhich express a specific antibody are incubated in the presence of acell which expresses the corresponding antigen, the bacteriophage willbind to the cell. Bacteriophage which do not express the antibody willnot bind to the cell. Such panning techniques are well known in the artand are described for example, in Wright et al. (supra).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Methods relating to productionof such display libraries, and the screening thereof, are set forth inU.S. Pat. No. 6,255,455, to Siegel, which is incorporated by referenceas if set forth in its entirety herein. Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors (or phagemids with M13 packaging signals) creating a library ofphage which express human Fab (or scFv) fragments on their surface.Phage which display the antibody of interest are selected by antigenbinding and are propagated in bacteria to produce soluble human Fab (orscFv) immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

Although the bacteriophage displaying antibodies of interest herein areexemplified by M13 phage, the present invention is not limited to these,or any other, vector displaying an antibody. Instead, one skilled in theart would appreciate, armed with the teachings provided herein, that anyvector that can display an antibody, wherein the vector comprises anucleic acid the sequence of which is at least partially known, can beused in the methods disclosed herein. Therefore, while theantibody-displaying bacteriophage disclosed herein are exemplified byM13, other bacteriophage, such as lambda phage or T7 phage, can also beuseful in the method of the invention. Lambda phage display librarieshave been generated which display peptides encoded by heterologous DNAon their surface (Sternberg et al., 1995, Proc. Natl. Acad. Sci. USA92:1609-1613) as have T7 phage display libraries (Hansen et al., 2001,Int. J. Oncol. 19:1303-1309).

Moreover, it is contemplated that the method of the invention may beextended to include viruses other than bacteriophage, such as eukaryoticviruses. In fact, eukaryotic viruses can be generated which encode genessuitable for delivery to a mammal and which encode and display anantibody capable of targeting a specific cell type or tissue into whichthe gene is to be delivered. For example, retroviral vectors have beengenerated which display functional antibody fragments (Russell et al.,1993, Nucl. Acids Res. 21:1081-1085). These, and any other vectorexpressing an antibody can be used in the methods of the invention andare encompassed thereby.

Furthermore, while the method of the invention as exemplified hereindescribes using phage which encode the Fab portion or an scFv portion ofan antibody molecule, the method should not be construed to be limitedsolely to the use of phage encoding Fab or scFv antibodies. Fabmolecules comprise the entire Ig light chain, that is, they compriseboth the variable and constant region of the light chain, but includeonly the variable region and first constant region domain (CH1) of theheavy chain. Single chain antibody molecules comprise a single chain ofprotein comprising the Ig Fv fragment. An Ig Fv fragment includes onlythe variable regions of the heavy and light chains of the antibody,having no constant region contained therein. Phage libraries comprisingscFv DNA may be generated following the procedures described in Marks etal., 1991, J. Mol. Biol. 222:581-597. Panning of phage so generated forthe isolation of a desired antibody is conducted as described herein forphage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities. Therefore, antibody-displaying libraries can be “natural”or “synthetic” (Barbas, 1995, Nature Medicine 1:837-839; de Kruifet al.,1995, J. Mol. Biol. 248:97-105). Antibody-displaying librariescomprising “natural” antibodies are generated as described in, e.g.,U.S. Pat. No. 5,876,925, to Siegel. Antibody-displaying librariescomprising “synthetic” antibodies are generated following the proceduredescribed in Barbas (1995, supra) and the references cited therein.

The skilled artisan would appreciate, based upon the disclosure providedherein, that the red blood cell antibodies to which antibodies can begenerated using methods known in the art and can then be used in themethod of the invention include, but are not limited to, Rh antigens,including Rh(D), Rh(C), Rh(c), Rh(E), Rh(e), and other non-Rh antigens,including red blood cell antigens in the Kell, Duffy, Lutheran and Kiddblood groups.

Thus, the method of the invention can be used for detection of any RBCantigen or other cell antigen, such as, but not limited to,tumor-specific antigen, bacterial antigens, and the like. The method ofthe invention is also useful for typing platelets by generating phageantibodies specific for a number of clinically important plateletantigens, notably, HPA-1a/1b, HPA-2a/2b, HPA-3a/3b, and the like.

The invention is further useful for typing donor white blood cells forHLA antigens for the purposes of matching donors and recipients forpotential transplant matching in the case of both solid (for example,kidney, heart, liver, lung) and non-solid (for example, bone marrow)organ or tissue transplanting.

In addition, the methods of the present invention can be used forforensic purposes, to detect any antigen of interest in a sample, wherethe sample can be, but is not limited to, bone, hair, skin, semen,saliva, or any other sample that can be obtained from an organism orbiological sample. The only feature required is that the sample containan antigen that can be specifically recognized by an antibody expressedby a bacteriophage, or other antibody-displaying vector. Thus, thepresent invention is not limited in any way to the detection of anyparticular antigen; instead, the invention encompasses detecting a wideplethora of antigens of interest using the novel“phenotyping-by-reagent-genotyping” detection methods disclosed herein.

Thus, the invention encompasses detecting an antigen of interest on ared blood cell, referred to herein as “phenotyping,” by detecting thebinding of a phage expressing an anti-red blood cell antibody, where thephage is detected by detecting a known sequence present in the nucleicacid contained by the phage particle, which is referred to herein as“phenotyping-by-reagent-genotyping.” Further, the invention includesscreening of patient sera for anti-red blood cell antibodies using phageparticles that display anti-human IgG (or anti-IgM or anti-kappa/lambdalight chain antibody which would pick up any Ig isotype). Again, thephage bound with the RBCs is detected by detecting a nucleic acidsequence present in the nucleic acid contained by the phage.

Additionally, the invention encompasses using thephenotyping-by-reagent-genotyping method in an immune assay, whether theantigen being detected is on a cell or not (e.g., antigens such as, butnot limited to, any measured for research or clinical purposes from acytokine to HCG for a pregnancy test). That is, the present inventioncombines the specificity conferred by immunoglobulins for a givensubstance, which specificity takes into account any post-translationalmodification (e.g., phosphorylation, glycosylation, and the like), withthe sensitivity conferred by nucleic acid detection methods—as well asthe ability to perform multiplex assays. That is, a sample being assayedwould be applied such that its components are affixed to a solidsupport, such as coating the well of a plate for an ELISA,nitrocellulose filter, bead, or any other solid support, and the phageexpressing a protein that specifically binds with a cognate ligand canbe allowed to bind with the components affixed to the solid support. Anyphage specifically bound to a cognate ligand can be detected bydetecting a known nucleic acid sequence specified by the nucleic acidcontained within the phage. Thus, the presence of any ligand of interestcan be detected using the “phenotyping-by-reagent-genotyping” methoddisclosed herein even where the sample being assayed does not comprise acell.

Moreover, the skilled artisan would appreciate, based upon thedisclosure provided herein, that the invention encompasses thephenotyping of other blood cells (e.g., platelets, white cells, and thelike) and the detection of antibodies to those cells in the blood (e.g.,anti-platelet auto- or alloantibodies, anti-HLA antibodies, etc.), suchthat the present invention is not limited to red blood cells. Indeed,the invention is not limited to blood cells at all, but can be used todetect any molecule of interest present on any kind of cell. Thus, oneskilled in the art would appreciate, based upon the disclosure providedherein, that the present invention includes, but is not limited to,detecting a molecule of interest on a cell where flow cytometry wouldotherwise be used such that the wide plethora of antibodies nowavailable (e.g., hundreds of anti-CD antibodies, such as anti-CD4 orCD-8 for helper/suppressor T cells, anti-CD20 for B cells, and the like)can be expressed on a phage and used to detect, according to the novelmethods disclosed elsewhere herein, whether the antigen is present in acell. The present invention includes using antibodies to be developed inthe future to antigens of interest as these are developed and usedaccording to the methods disclosed herein.

The skilled artisan would appreciate, based upon the teachings providedherein, that detection of any molecule of interest, for instance, withregard to forensic application of the methods disclosed herein, providesan important advantage over present methods in that many antigensimportant for identifying the origin of fluids (blood or solublesubstances in saliva, and the like) are carbohydrates (like the A and Bantigens). Using genetic testing on the miniscule spot for DNA cannotamplify the DNA that encodes carbohydrates because DNA does not encodecarbohydrates which are products of post-translational modification ofproteins. Prior art methods relating to carbohydrate detection arelimited to detecting the DNA for the enzymes (e.g., theglycosytransferases) that are responsible for assembling the sugarmoieties onto the protein or lipid. The problem with conventionaldetection assays is that the ultimate expression of a particular sugaris the result of the inheritance of a number of enzymes that act inprecise sequence to assemble the chains such that the genes for all ofthe enzymes would need to be detected in order to identify the identityof the person the sample was derived from. For example, in order for anindividual to be blood group A, the enzyme that addsN-acetylgalactosamine onto its precursor sugar is required, as is theenzyme (a fucosyl transferase) to assemble the precursor sugar. Othercarbohydrates (like P) are even more complicated in their structures andassembly. If the sample comprises a mixture of secretions in one spotfrom different individuals, DNA testing would pick up all enzymes andthe test would not be able to distinguish whether one person had all theenzymes and could make a particular sugar antigen or if the samplecomprised DNA from various persons who could each only produce thevarious sugar components. Unlike conventional nucleic acid-basedtesting, the present invention provides the advantage of combining theexquisite specificity of an antibody that is capable of recognizing acomplex structure, such as a glycan, and the ability to detect minisculequantities of a nucleic acid; thus, detection of the nucleic acidcontained by the phage, combined with the specificity of an antibody,provide a novel assay with the extraordinary sensitivity and specificityrequired in forensic uses.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that while the term “phenotyping” is generally used in theart to detecting a characteristic demonstrated by a cell, or organism,the term as used herein with regard to“phenotyping-by-reagent-genotyping”, relates to the identification ofany antigen of interest, whether or not the antigen is associated with acell, by detecting a nucleic acid sequence. Thus, for instance, theidentification of a drug in a dried spot on a car door using aphage-displayed anti-drug antibody according to the methods of theinvention, would be “phenotyping” as the term is used herein. Therefore,the methods of the invention, where an antibody expressed by a phagebinds with a cognate antigen and the antigen is detected by assaying fora nucleic acid sequence present in the phage DNA, is “phenotyping” asused herein.

Indeed, the skilled artisan, armed with the teachings provided herein,would realize that the present invention is not limited to detection ofan “antigen” using phage-displayed antibody (which antibody is thendetected by detecting a nucleic acid sequence encoded by the phage DNA).Instead, the present invention encompasses using a non-antibody proteinexpressed by a phage, which protein specifically binds with a cognateligand present on a cell, in a sample, or both. Many such binding pairsare well-known in the art and have been identified using a wide varietyof assays, including yeast two- and three-hybrid binding assays, among awide plethora of other assays. Thus, where a binding pair is known inthe art, one of the two molecules can be expressed by the phage (thebinding pair protein expressed by the phage is referred to herein as the“receptor”) and the presence of the other member of the binding pair(referred to as the “ligand” or “target”) can be detected by detecting anucleic acid sequence contained by the phage expressing the receptorprotein. The ligand that is to be detected by its cognatereceptor/binding partner expressed by the phage can include, but is notlimited to, a hormone, or a portion of a hormone where the portion canbind with the receptor displayed by the phage. Further, the methods ofthe present invention can be used to, inter alia, measure the expressionof a hormone receptor on a cell by assessing the amount of a phagedisplaying the hormone, or portion thereof, which binds with the cellbeing assayed. The phage specifically bound with the cell due to thereceptor/ligand (hormone receptor/hormone expressed by the phage,respectively) interaction can be detected by detecting a nucleic acidsequence present in the nucleic acid contained by the phage as morefully disclosed elsewhere herein.

One skilled in the art would understand, based upon the disclosureprovided herein, that the present invention encompasses detection of amolecule of interest that is not associated with a cell. That is, thepresent invention includes assaying for the presence of a molecule ofinterest in any sample where the sample can be applied to a solidsupport such that the molecule of interest can be immobilized. A phageexpressing an receptor known to bind specifically with that molecule(herein referred to as a “ligand” or “target” molecule) can then becontacted with the immobilized sample and the binding of any phage canbe detected by assaying for the presence of a nucleic acid sequencecontained by the phage as more fully described elsewhere wherein. Inthis way, the present invention can be used to detect a molecule ofinterest (ligand) present in any sample using the“phenotyping-by-reagent-genotyping” methods disclosed herein.

The skilled artisan would also appreciate, based upon the disclosureprovided herein, that a phage can readily expresses a peptide that isknown to detect cancer cells but where it is not known what component onthe cancer cell the peptide binds with. Thus, the protein known to bindcancer cells can be used to detect a cancer cell even though theidentity of the ligand/binding partner that binds with the protein isnot known, by detecting bound phage by detecting a nucleic acid sequencecontained by the phage, all as more fully disclosed elsewhere herein.

Additionally, where the phage is used to detect the binding of serumantibodies to a reagent red blood cell, the phage can express StaphProtein A, or a portion thereof, instead of anti-IgG, to detectimmunoglobulins bound with the RBCs. Therefore, the skilled artisanwould appreciate, based upon the disclosure provided herein, that a wideplethora of molecules can be expressed by the phage to detect a cognatebinding partner present on a cell, in a tissue or aqueous sample, andthe like, and the present invention is not in any way limited to phageexpressing an antibody, or to detection of an antigen on a cell, asexemplified elsewhere herein. That is, once a binding pair is known, theskilled artisan, armed with the disclosure provided herein, wouldreadily be able to detect one of the binding pair using the methods ofthe invention, i.e., by expressing one member of the binding pair on aphage and contacting the phage with a sample, then detecting any phagespecifically bound with the sample by detecting a nucleic acid sequenceencoded by the phage nucleic acid. This allows the rapid and sensitivedetection of a molecule of interest, or various molecules of interestwhere multiplexing is used, where the molecule is not a nucleic acid, bydetecting a nucleic acid.

The specific conditions under which the antibody, or receptor, displayedby the bacteriophage is allowed to specifically bind with an antigen, orligand, of interest will depend on the specific antigen-antibody and/orreceptor-ligand complex involved in the reaction. The skilled artisanwould understand, based upon the disclosure provided herein, that suchconditions can be readily determined for each antigen/binding pair beingdetected and the antibody/receptor being used to do so, as isexemplified herein for detection of Rh(D) and B antigens on intact redblood cells using phage expressing antibodies specific for theseantigens. These techniques for determining binding conditions areroutinely practiced in the art, and are therefore not described furtherherein.

Once the bacteriophage expressing the antibody (or receptor) arespecifically bound with the cell, or ligand in a sample, via theinteraction between the antigen-bearing moiety on a molecule of interestpresent on the cell (ligand) and the antibody expressed by the phage(receptor), the presence of bound phage is detected by detecting thenucleic acid contained in the bacteriophage particle. For the M13 phageexemplified herein, the nucleic acid is a single-stranded DNA molecule,but the present invention is not limited to any particular nucleic acid;rather, any nucleic acid can be detected using techniques well-known inthe art (e.g., as described in Sambrook et al., 1989, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York; andAusubel et al., 1997, Current Protocols in Molecular Biology, John Wiley& Sons, New York), some of which are disclosed herein, as well astechniques to be developed in the future, and these various techniquesare all encompassed in the invention.

The present invention also encompasses amplification of the nucleic acidto assist in its detection. However, the present invention is notlimited to methods requiring the amplification of the nucleic acid.Instead, the skilled artisan, based upon the disclosure provided herein,would appreciate that detection methods which do not requireamplification of the nucleic acid are encompassed in the invention. Suchdetection methods include, but are not limited to, detection of anucleic acid directly transferred to a chip wherein a fluorescent (orenzyme)-labeled oligonucleotide complementary to the phage(mid) sequencecan detect the unamplified nucleic acid. Thus, while FIG. 1 isillustrative of the various techniques that can be used to detect thenucleic acid sequence of interest, the invention is not limited toprocedures that require amplification prior to detection of thesequence. Therefore, PCR, IDAT, or other amplification reactions arepreferred, but not required, to practice the invention.

The skilled artisan would understand, once armed with the teachingsprovided herein, that, as exemplified herein, the nucleic acid can beamplified using convention polymerase chain reaction assays. That is, aset of primer sequences can be developed based on the known sequence ofthe nucleic acid contained by the bacteriophage. As discussed elsewhereherein, the primers can be specific for any portion of the nucleic acid,either the unique sequence comprised in the portion of the nucleic acidencoding the CDR3 portion of the antibody, or any other sequence presentin the nucleic acid. Thus, one primer can be complementary to a genericsequence contained in the phage DNA (irrespective of antibodyspecificity) and the other primer can be complementary to, e.g., asequence specific to that phage, such as, but not limited to, the CDR3hypervariable region of the antibody's heavy chain (i.e., the sequencethat is unique for a given antibody).

Detection of the amplified nucleic acid indicates the presence of theantigen recognized by the specific antibody encoded by thebacteriophage. The production of PCR primers, and probes that hybridizewith the sequence amplified by the PCR, are well-known in the art, andthese methods are described in, among others, Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York), and Ausubel et al. (1997, Current Protocols in MolecularBiology, John Wiley & Sons, New York).

Additionally, the skilled artisan would appreciate, based upon thedisclosure provided herein, that sequences can be inserted into thenucleic acid encoding the antibody expressed by the bacteriophage, whichinserted sequence can then be detected using various assays known in theart. For instance, as discussed elsewhere herein, “molecular beacons”,or as used herein, “beacons” or “beacon sequences,” arestem-and-loop-structured oligonucleotides with a fluorescent label atthe 5′ end and a universal quencher at the 3′ end (see, e.g., Tyagi andKramer, 1996, Nature Biotech. 14:303-308; Broude, 2002, Trends inBiotechnology 20:249-256). When the stem is closed (in the absence ofcomplementary nucleic acid), the fluorophore and quencher are in closeproximity and fluorescent energy is absorbed by the quencher andfluorescence is quenched and not detectable. In the presence ofcomplementary nucleic acid, the loop of the beacon hybridizes and thefluorophore and quencher separate such that quenching does not occur.Photons are then emitted from the fluorophore, unquenched, at thewavelength specific for that fluorophore and fluorescence is thendetectable. By combining a number of beacons in one tube, each with adifferent fluorophore at their 5′ ends, multiple DNA (Tyagi et al, 1998,Nature Biotech. 16:49-53) or RNA (de Baar et al., 2001, J. Clin.Microbiol. 39:1895-1902) targets can be simultaneously detected bymeasuring the spectrum of colors emitted from the reaction vessel.

Molecular beacons of two, or more, different colors can be incorporatedinto a PCR and/or a transcription reaction (e.g., IDAT) to detect thepresence of antibody-specific DNA. As described elsewhere herein, thenucleic acid of each bacteriophage, encoding an antibody specific for anantigen of interest, can be modified to insert a unique beacon sequenceand each molecular beacon probe can be conjugated to a uniquequencher/fluorophore pair such that each beacon, when bound with itscomplementary sequence, will fluoresce at a unique frequency. In thisway, each beacon can be used to detect an antibody binding with anantigen such that the “multiplex” reaction can yield resultsdemonstrating which antigens are present on a cell being examined byassessing which fluorophores are present in the sample. The design andproduction of such “beacon” sequences, and nucleic acid sequencescomprising sequences complementary thereto, are well known in the art.

Armed with the disclosure provided herein, the skilled artisan wouldunderstand that the present invention is not limited in the number ofmolecules of interest that can be detected in a single multiplexreaction. That is, the design of unique sequences that can be detectedand distinguished from the each other in a single reaction is well-knownin the art. Further, one skilled in the art would appreciate, based uponthe disclosure provided herein, that various technologies, such as, butnot limited to, microchip arrays, slot blots, use of beacon probes, andother high-throughput assays allowing the processing of many samples,and providing the capability for multiplex assays, can be used in themethods of the present invention as exemplified herein, as known in theart, or using techniques to be developed in the future, the use of whichcan be readily contemplated based upon the disclosure provided herein.That is, current chip technology already provides that the number ofantigens that can be assayed on a single chip exceeds the number ofknown red blood cell antigens. Further, where the cycling parameters ofvarious PCR reactions are compatible, a single tube comprising numerousprimer pairs can be used to multiplex the PCR reactions. Thus,multiplexing the reactions relating to the methods of the inventionwould appear to only be limited as to the number of spots on the chips,since the binding of phage to cells, the number of primers that can beused perform PCR in a single tube, and the like, do not limit the numbermolecules that can be assayed for using the methods of the invention.

The skilled artisan would understand, based upon the disclosure providedherein, that the invention encompasses amplification of the nucleic acidof interest (i.e., the nucleic acid contained by the bacteriophageexpressing the antibody to the antigen-bearing moiety of interest whichis bound to the cell by the specific binding of the antibody with itscognate antigen) using any method known in the art, as well as methodsto be developed in the future. PCR amplification was discussedpreviously herein, and is exemplified elsewhere herein, as is IDAT,which is amplification of the nucleic acid using a transcription-basedmethod. However, these are exemplary amplification methods only, and thepresent invention is not in any way limited to these, or any other,method for amplifying the nucleic acid contained by the phage ofinterest.

The invention also encompasses detecting the nucleic acid once it hasbeen amplified. One skilled in the art would appreciate, once armed withthe teachings provided herein, that any method for detection of anucleic acid known in the art, or to be developed in the future, can beused to detect the nucleic acid in the method of the invention. Suchdetection methods include, but are not limited to, real-time PCR usingfluorescent probes, detecting amplicons of the predicted size using sizeseparation techniques (e.g., agarose gel electrophoresis), Southern andNorthern blotting techniques, hybridization to oligonucleotidemicroarrays, and use of “molecular beacon” probes, discussed more fullyelsewhere herein. Further, as more fully disclosed elsewhere herein,techniques to automate, accelerate, or otherwise improve the detectionof the nucleic acid sequence of interest are contemplated. Suchtechniques include, but are not limited to, “electric field-acceleratedhybridization to oligonucleotide microarrays” (Su et al., 2002,Electrophoresis 23:1551-1557), which provides rapid results, e.g., timefrom application of DNA (or RNA) to readout is less than about 10minutes. Thus, techniques to improve the efficiency of the detectionstep are encompassed in the invention as would be understood by theskilled artisan.

B. Detection of Multiple Antigens

The present invention encompasses a method for detecting the presence ofat least two different antigen-bearing moieties on a cell. The methodcomprises contacting at least two different bacteriophage, each encodingand expressing an antibody that specifically binds an antigen, where thetwo antibodies do not bind the same antigen. Any phage that arenon-specifically bound with the cell are removed (e.g., by washing thecell), and the presence of any bound bacteriophage is detected bydetecting the nucleic acid present in the phage. That is, are more fullydescribed elsewhere herein, the sequence, or a portion thereof, of thenucleic acid present in the phage particle is exposed and the presenceof the nucleic acid (i.e., the presence of its known nucleic acidsequence) is detected using methods well-known in the art. Because eachbacteriophage comprises a nucleic acid sequence that is distinguishablefrom those present in other bacteriophages present in the same sample,the presence of various antigens can be detected in a single samplemixture. Such “multiplex” assays are not possible using antibody-baseddetection methods, since the reagents used to detect the presence ofantibodies bound with the cell cannot readily distinguish between eachantibody. Further, conventional blood typing does not use reagents thatdetect the presence of antibodies bound with the cell since many bloodtyping reagents, typically the decavalent IgMs, directly agglutinate thecells. In those assays, one cannot multiplex the reaction it would notbe possible to determine which reagent caused the agglutination.However, methods based on detecting multiple, unique nucleic acidsequences, make assaying for various antigens, by detecting the nucleicacid sequences present within phage particles bound to/linked with thoseantigens via an antibody molecule expressed by the phage, possible asdemonstrated herein.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the various bacteriophage, each displaying adifferent antibody recognizing an antigen distinct from the antigensrecognized by any other phage-displayed antibody present in the sample,can be contacted with the cell being assayed simultaneously, in the samereaction mixture. However, the bacteriophage can be contacted with thecell in serial fashion, such that each bacteriophage contacted with thecell, any unbound bacteriophage is removed, and the next bacteriophagecan be contacted with the cell, the unbound phage removed, and on andon, until all of the bacteriophage have been allowed to bind with thecell such that all of the antigens of interest have been assayed for onthe cell. All the bound phage can then be treated to release the nucleicacids present within, and the various nucleic acid sequences present inthe sample can be detected as discussed more fully elsewhere herein.Because each bacteriophage expressing a unique antibody contains anucleic acid comprising a known sequence that is distinct from thesequences of all the other bacteriophage nucleic acids used in theassay, the binding of each bacteriophage can be determined separatelyfrom all the others. Thus, the presence of each antigen assayed for canbe determined by detecting the unique nucleic acid sequence associatedwith the bacteriophage displaying the antibody that bound with thatantigen because detecting various nucleic acid sequences in a sampledoes not interfere with the detection of other, unrelated, sequences inthat same sample.

The skilled artisan would appreciate, based upon the disclosure providedherein, that where speed is desired, different antigens can be assayedfor in a single reaction mixture. Moreover, where greater sensitivity ofthe assay is desired, e.g., where forensic detection of a small sampleis involved, or where the particular combination of phage required forthe assay are somehow incompatible with the same amplification scheme orconditions, then the various reactions can be performed serially. Thus,while it is preferred that PCR be performed by adding all the relevantprimers into one tube and amplifying all the fragments at once, theinvention also encompasses methods where each antigen/ligand isidentified in serial fashion using the same sample.

In designing the primers and the stretches of phage(mid) DNA to amplify.it is therefore preferable to design specific sequences (tags) to beamplified in the phage DNA, rather than exploiting the difference inantibody or peptide sequence, since one can make them compatible interms of multiplexing and cycling conditions. As exemplified herein fordetection of B and Rh(D) antigens on an RBC using anti-B and anti-Rb(D)displayed by phage, the primers can be designed to be used in a singlereaction and the phage were added together to the RBCs and the PCR wasperformed in a single tube to produce both 1100 bp and 1600 bpamplicons. While this is one method, the invention is not limited tothis particular scheme.

The present invention also features a method for the detection of phageDNA in the process of detection of multiple antigens by conducting a PCRreaction using a set of nucleic acid primers that anneal to common siteswithin all phage antibody DNAs. In this embodiment, the length and/orcomposition of DNA between the common primers differs between thedifferent phage DNAs. Differences in the phage DNAs are detected bydetermining the melting temperature of the PCR products.

In an embodiment of the invention, differences in the phage DNAs aredetected by using a capillary PCR fluorescent device. The PCR reactionis conducted in the presence of a dye that binds to double stranded DNA.In one aspect of the invention, the dye is SYBR GREEN I. After the PCRreaction has been conducted, the melting temperatures of the PCRproducts are determined using a PCR fluorescent device, such as, but notlimited to, a capillary PCR fluorescent device. A melting temperatureprofile is then generated which indicates the melting temperatures ofthe PCR products. By analyzing the melting temperature profile, thespecific phage DNA amplified by the PCR reaction can be determined,thereby identifying the specific phage antibody bound to the cell.

Therefore, a number of different phage-displayed antibodies (e.g.,antibodies specific for various blood group antigens) can be contactedsimultaneously with a sample of RBCs. The unbound phage are removed, andthe nucleic acids of the phage bound with the cells are assayed todetermine which phage bound with the cells. Since each bacteriophagecontains a unique sequence “tag”, nucleic acid methods can be used todetermine which phage, and therefore, which antigens, are present on thecells. This “multiplex” method is a vast improvement over prior artmethods which require that each antigen be assayed for separately,thereby requiring additional reagents, increasing the technicaldifficulty and length of the assay, and introducing more opportunity forerrors in requiring additional steps and manipulations.

Accordingly, a number of different phage-displayed blood groupantibodies can be contacted simultaneously to the same sample of redcells and the differences in antibody nucleotide sequence can beexploited to determine which ones bound and which ones did not, asdemonstrated herein using anti B and anti-Rh(D) antibodies displayed ondifferent phage. Such “multiplexing” is not possible by agglutinationmethods as one could never tell which antibody(ies) caused theagglutination.

That such a methodology is possible, i.e., that the simultaneous bindingof multiple anti-RBC antibodies can be detected by the amplification anddetection of antibody DNA, is demonstrated by the data disclosed hereinwhere a model system comprising phage-displayed anti-blood group B andanti-Rh(D) human monoclonal antibodies was employed. However, theskilled artisan, based upon the disclosure provided herein, wouldreadily appreciate that such “multiplexing” strategy is not limited toany particular antibodies, but can be used to detect multiple red bloodcell antigens using a wide plethora of antibody-displaying phage, whereeach phage comprises a DNA sequence that can be detectably distinguishedfrom the nucleic acid of other phage encoding antibodies havingdifferent specificities, or even phage encoding antibodies having thesame specificities, so long as the nucleic acids of the phage can bedistinguished from one another. Indeed, these methods are not limited tored blood cells or their antigens, but can be readily applied to anysystem where it is desirable to detect the presence of multiple antigenson a cell, or in a sample.

The skilled artisan would appreciate, as more fully discussed elsewhereherein, that where several antibody-displaying phage, each reactive witha different antigen of interest, can be used in a “multiplex” reactionwhere the antigens are detected in a single reaction, and/or within thesame sample, the primers are selected such that the regions amplified byeach primer pair (i.e., forward and reverse primers and, if desired,probe for the amplicon produced therefrom) are each distinguishable fromeach other.

C. Detection of Antibody in Serum

The present invention includes a method for detecting the presence ofautoantibodies or alloantibodies in serum, more specifically, fordetecting anti-red blood cell antibodies present in human serum(indirect antiglobulin test). The method comprises contacting a humanred blood cell expressing at least one red blood cell antigen with aserum sample to be assayed. The cell is washed to removenon-specifically bound antibodies and the cell is then contacted withbacteriophage displaying an antiglobulin reagent on its surface. Wherethere is a human antibody (IgG, IgM, and the like) bound with the cell,the bacteriophage will bind via the antiglobulin reagent displayed bythe phage. The presence of phage specifically bound with the cell (viabinding with the human antibody on the cell) can then be detected asdisclosed herein based on detection of a known nucleic acid sequencepresent in the bacteriophage. In this way, where the antigen compositionof a panel of cells is known, this reference panel of cells can be usedto assay for the presence of antibodies to these antigens in any sampleby simply and rapidly detecting the nucleic acid of a bacteriophagedisplaying an antiglobulin on its surface, such that“phenotyping-by-genotyping” can be used to increase the efficiency andsensitivity, as well as to automate, assays that were previouslyperformed using antibody-based detection methods.

D. Detection of Antibody or Complement Fragments on Red Blood Cells

The present invention includes a method for detecting the presence ofautoantibodies, alloantibodies, or complement fragments bound to thesurface of red blood cells, more specifically, for the diagnosis ofautoimmune hemolytic anemia or for the determination of alloimmunedestruction of transfused red blood cells (direct antiglobulin test).The method comprises washing a sample of red blood cells to removenon-specifically bound antibodies and then contacting the cells withbacteriophage displaying an antiglobulin reagent on its surface. Wherethere is human antibody or complement bound with the cell, thebacteriophage will bind via the antiglobulin reagent displayed by thephage. The presence of phage specifically bound with the cell (viabinding with the human antibody or complement on the cell) can then bedetected as disclosed herein based on detection of a known nucleic acidsequence present in the bacteriophage. In this way,“phenotyping-by-genotyping” can be used to increase the efficiency andsensitivity, as well as to automate, assays that were previouslyperformed using antibody-based detection methods.

E. Performing Donor/Recipient Compatibility Testing

The present invention includes a method for assuring compatibility,i.e., non-reactivity, between antibodies in patient sera and an aliquotof red blood cells drawn from a unit of blood intended for transfusion(crossmatching). The method comprises contacting a sample ofcharacterized donor red blood cells with a patient serum sample to betested. The cells are washed to remove non-specifically bound antibodiesand the cell is then contacted with bacteriophage displaying anantiglobulin reagent on its surface. Where there is human antibody boundwith the cell, such as would be the case with an incompatiblecrossmatch, the bacteriophage will bind via the antiglobulin reagentdisplayed by the phage. The presence of phage specifically bound withthe cell (via binding with the human antibody on the cell) can then bedetected as disclosed herein based on detection of a known nucleic acidsequence present in the bacteriophage. In this way,“phenotyping-by-genotyping” can be used to increase the efficiency andsensitivity, as well as to automate, assays that were previouslyperformed using antibody-based detection methods.

II. Kits

The invention includes various kits which comprise a compound, such as abacteriophage displaying an antibody with known specificity for anantigen of interest, a primer pair for amplifying a known nucleic acidsequence present in the phage, a molecular beacon for detecting a knownsequence present in the nucleic acid contained in the bacteriophage, areagent for use in an IDAT reaction (e.g., T7 RNA polymerase, DNApolymerase I, dNTPs, and the like), and/or compositions of theinvention, an applicator, and instructional materials which describe useof the compound to perform the methods of the invention, and anycombination of the preceding components. Although exemplary kits aredescribed below, the contents of other useful kits will be apparent tothe skilled artisan in light of the present disclosure. Each of thesekits is included within the invention.

In one aspect, the invention includes a kit for detecting the presenceof an antigen-bearing moiety on a cell. The kit is used pursuant to themethods disclosed in the invention. Briefly, the kit may be used tocontact a bacteriophage displaying an antibody that specifically bindswith the antigen-bearing moiety when it is present on a cell. This isbecause, as more fully disclosed elsewhere herein, binding of thebacteriophage with the cell, and subsequent detection of a nucleic acidsequence known to be present in the phage, indicates that the phagebound with the cell, thereby indicating that the antibody displayed bythe phage bound with its cognate antigen, thus, in turn, indicating thatthe antigen is present on the cell, thereby detecting the antigen bythis novel “phenotyping-by-genotyping” method of the invention.

The kit further comprises an applicator useful for administering thebacteriophage, PCR primers, molecular beacons, and the like, to asample. The particular applicator included in the kit will depend on,e.g., the method used to detect the antigen using“phenotyping-by-genotyping” as disclosed herein, and such applicatorsare well-known in the art and may include, among other things, apipette, a syringe, a dropper, and the like. Moreover, the kit comprisesan instructional material for the use of the kit. These instructionssimply embody the disclosure provided herein.

In one aspect, the kit further comprises a bacteriophage expressing anantibody that specifically binds a red blood cell antigen, such as, butnot limited to, RBC antigens A, B, Rh(D), Rh(C), Rh(c), Rh(E), Rh(e), K,Fy^(a), Fy^(b), M, N, S, s, Jk^(a), Jk^(b).

Further, in another aspect, the kit further comprises a molecular beaconprobe wherein the nucleic acid sequence of the probe is complementarywith a sequence such as, for instance, of the sequence of SEQ ID NO:3and the sequence of SEQ ID NO:4, as exemplified herein. These sequencesare contained within the nucleic acid contained by the bacteriophagesuch that sequences hybridizing therewith can detect the presence ofphage(mid) nucleic acid. More specifically, the kit comprises amolecular beacon probe having a sequence such as, but not limited to,the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

In yet another aspect, the kit comprises a PCR primer than can amplifythe nucleic acid sequence present in the phage. Such a PCR primerincludes, but is not limited to, a primer comprising the sequence of SEQID NO:1 and the sequence of SEQ ID NO:2.

The kit includes a pharmaceutically-acceptable carrier. The compositionis provided in an appropriate amount as set forth elsewhere herein.

Additional kits, such as those for detecting complement, and auto- andallo-antibodies in a sample, as well as kits for detecting any ligand ofinterest where a known ligand/receptor binding pair is known, are alsoincluded as would be readily appreciated by one skilled in the art basedupon the disclosure provided herein.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

EXAMPLES

Current technologies used in blood collection facilities, blood banks,and transfusion service laboratories are extraordinarily laborintensive, prone to human error, and an order of magnitude moreexpensive per test that those in other clinical laboratories. Coupledwith a growing shortage of skilled medical technologists, dwindlingsupplies of human plasma-derived phenotyping reagents, and an inherentdifficulty in fully automating 1950's-based agglutination methodologies,the ability to perform the hundreds of millions of pre-transfusion testsper year in a rapid, accurate, and cost-effective manner is asignificant challenge.

The present invention relates to the development of novel moleculartechnologies and reagents pertinent thereto, to develop a new class ofrenewable, inexpensive, high-quality blood bank testing reagents thatfunction in a rapid, high-throughput, automatable assay system.

A central feature of the novel technologies disclosed herein are redblood cell antigen-specific monoclonal antibodies displayed on thesurface of bacteriophage particles. The naturally-occurring presence ofunique DNA sequences within the phage particles has been exploited todevelop an assay system in which the phenotype of a red cell isdetermined by assaying the genotype of the detecting reagent, i.e., thephage bearing an antibody that specifically binds with an antigenpresent on a red blood cell.

Such a strategy offers extraordinary sensitivity and specificity,requires minute amounts of testing materials and reagents, is easilyadapted to automation, and is amenable to multiplexing strategiesthereby offering the ability to perform simultaneous antigen profilingof a red cell sample in a single reaction vessel, all which offerssubstantial improvement over prior art methods.

A panel of phage-displayed antibody reagents specific forclinically-significant red cell antigens is developed using antibodyphage display library technologies. Examples of these reagents andmethodologies for their production have described previously (see, e.g.,U.S. Pat. Nos. 5,876,925, and 6,255,455, both of which are incorporatedby reference herein in their entirety), and are exemplified by thereagents used herein. These phage-display antibody reagents have beendemonstrated to be superior to conventional blood bank reagents and canbe used with all currently-available agglutination-based blood typingmethods. Moreover, a novel blood typing platform based on this newgeneration of anti-red blood cell antibodies is disclosed, which novelplatform makes full use of the coupled phenotypic/genotypic propertiesof these novel reagents.

Thus, the data disclosed herein demonstrates that the present inventionovercomes several long-standing technical hurdles in the field of bloodtyping. The data disclosed herein demonstrate development of a new classof renewable, inexpensive, high-quality blood bank testing reagents andmethodologies pertaining thereto, that function in a rapid,high-throughput, automatable assay system.

A feature of the novel technology disclosed herein are RBCantigen-specific monoclonal antibodies displayed on the surface offilamentous phage particles that are isolated using a number oftechnologies well-known in the art, and such technologies as aredeveloped in the future. The phage particles physically link thephenotype of an antibody displayed on the phage (the antigen-bindingmoiety) with its genotype (the unique sequence of DNA within theparticle that encodes the amino acid sequence of that particularantigen-binding moiety). Additionally, the phage particle can link thephenotype of the antibody displayed on its surface and the DNA presentin the phage particle in that another portion of the DNA, which does notencode the antigen-binding portion of the molecule but which isassociated therewith (i.e., a beacon sequence), can be detected suchthat detecting the identity of the antigen bound by the antibodydisplayed on the phage can be readily determined by detecting thepresence of the beacon.

Thus, the naturally-occurring presence of unique DNA sequences withinthe particles has been exploited herein by developing a novel assaysystem in which the phenotype of an RBC being assayed is determined byassaying the genotype of the detecting reagent, i.e., theantibody-displaying phage and the DNA molecule encoding such antibody,or another unique DNA sequence (i.e., a beacon sequence) within the DNAcontained by the phage. The rationale behind the development of thisnovel “phenotyping-by-reagent-genotyping” approach is the recognitionthat methodologies which use nucleic acid detection schemes offer thehighest sensitivity and specificity, require minute amounts of testingmaterials and reagents, and are readily adaptable to automation.

Furthermore, nucleic acid-based assays are amenable to multiplexingstrategies which, in the case of blood typing, would offer thepossibility of simultaneously determining the antigen profile of a givenRBC sample in a single reaction vessel. The ability to multiplex typingreactions using the technology proposed in this research applicationwould represent a significant advantage for both blood collectionfacilities and transfusion services which historically have been limitedto the conventional “one tube/one result” agglutination methodology.Therefore, the novel assays described herein allow the detection ofmultiple antigen-bearing moieties present on an RBC to be readily andquickly detected.

Phage-Display Technology

At the core of the proposed technology are RBC antigen-specificmonoclonal antibodies which are displayed on the surface of filamentousbacteriophage particles (reviewed in Siegel, 2001, Transfusion Med. Rev.15:35-52). In contrast to expensive and time-consuming conventionalcellular methods for generating monoclonal antibodies fromB-lymphocytes, antibody phage display works by immortalizing theimmunoglobulin genes rather than the cells from which they were derived.By using molecular methods instead of cell transformation, “libraries”of phage particles are produced from populations of B-cells, eachparticle displaying a particular antibody specificity on the outside andcontaining the antibody's unique DNA sequence on the inside.

Methods for selecting phage particles specific to particularcell-surface antigens from such libraries have been described previously(e.g., Siegel et al., 1997, J. Immunol. Meth. 206:73-85; U.S. Pat. No.5,876,925, to Siegel) and hundreds of unique human anti-Rh(D) monoclonalphage-displayed antibodies have been produced to date (e.g., Siegel etal., 1997, J. Immunol. Meth. 206:73-85; Chang and Siegel, 1998, Blood91:3066-3078; U.S. Pat. No. 6,255,455, to Siegel). Although monoclonalantibodies produced in this way can be expressed as soluble antibodymolecules (unlinked to phage) that can agglutinate RBCs using theconventional indirect antiglobulin (i.e., Coombs) reaction (see Siegeland Silberstein, 1994, Blood 83:2334-2344), it has been established thatthe actual phage particles displaying the recombinant monoclonalantibodies can be used in agglutination reactions by substitutinganti-M13 phage antibody for the Coombs reagent (Siegel et al., 1997, J.Immunol. Meth. 206:73-85; U.S. Pat. No. 5,985,543, to Siegel). Anadvantage of this method in agglutination assays using intact phagedisplaying the antibody is increased sensitivity since as few asapproximately 10 anti-Rh(D)-expressing phage particles (compare withabout 150-1000 conventional IgG) are needed to induce agglutination dueto the greater degree of crosslinking by anti-M13 afforded by therelatively large size (approximately 0.5 microns) of the particles.

More importantly, for commercial application, is the ability of suchphage-displayed antibodies to direct their own replication within E.coli, allowing enough reagent to be produced for use in conventional redcell typing of nearly 500,000 units of blood for a reagent cost of a fewdollars (see Siegel et al., 1997, J. Immunol. Meth. 206:73-85).

The substitution of conventional blood bank typing reagents withphage-displayed recombinant antibodies in agglutination assays is a vastimprovement over prior art Coombs-based agglutination methodologies inand of itself for the reasons stated above—the ability to clone humanantibodies without the need to B-cell transformation, greater assaysensitivity, inexpensive production in bacterial culture, and others(Siegel, 2001, Transfusion Med. Rev. 15:35-52). However, the datadisclosed herein demonstrate the further dramatic improvement upon thephage-based technology by exploiting the naturally-occurring presence ofunique DNA sequences contained within the antibody-expressing phageparticles to facilitate high-throughput automation and multiple-antigentyping in a single reaction vessel (multiplexing). The method of theinvention can comprise the various steps illustrated in FIG. 1, and ismore fully disclosed elsewhere herein.

Using antibody phage-display and other technologies available in theart, a set of novel monoclonal reagents specific forclinically-significant RBC antigens can be cloned, produced, and theperformance characteristics thereof can be validate according to theteachings provided herein, as well as methods known in the art and to bedeveloped in the future. For instance, previous studies demonstrated theproduction and isolation of such reagents with specificities for RBCantigens B, anti-Rh(D), M and N (see, e.g., Chang and Siegel, 2001,Transfusion. 41:6-12; Siegel et al., 1997, J. Immunol. Meth. 206:73-85;Chang and Siegel, 1998, Blood 91:3066-3078; Czerwinski et al., 1995,Transfusion. 35:137-144; Czerwinski et al., 1999, Transfusion.39:364-371). Such methods can be applied to develop, among others,anti-A, anti-Rh(C, c, E, e), as well as antibodies in the Kell, Duffy,Kidd, and Ss blood groups. These reagents can be used in conventionalmanual and automated agglutination assays, as well as in the novelmethods disclosed herein.

An index set of anti-blood group B and anti-Rh(D) phage was produced andunique DNA sequence tags (i.e., beacon sequences), oligonucleotideprimer and hybridization sites, and polymerase promoters are insertedinto the DNA that codes for each antibody. The performancecharacteristics of a number of nucleic acid amplification/detectionschemes is assessed to identify and quantify the RBC binding of eachreagent as exemplified herein using group B and anti-Rh(D) phagereagents.

The data disclosed herein demonstrate that polymerase chain reaction(PCR) and agarose gel electrophoresis can be used to simultaneouslydetect and differentiate the binding of two different anti-RBC antibodyspecificities. These data demonstrate that screening using these methodscan be performed rapidly, and can be scaled, and automated forcommercial application.

Amplification of Phase DNA Using the Polymerase Chain Reaction:

In one aspect, the binding of a RBC-specific phage-displayed antibody,e.g., a phage particle expressing anti-Rh(D), was detected through theaddition of oligonucleotide primers specific to the anti-Rh(D)'s nucleicacid sequence exposed when, for example, the bound phage particles wereheated to denature the phage coat. One primer can be complementary to ageneric sequence contained in the phage DNA (irrespective of antibodyspecificity) and the other primer can be complementary to, e.g., asequence specific to that phage, such as, but not limited to, the CDR3hypervariable region of the antibody's heavy chain (i.e., the sequencethat is unique for a given antibody). The measurement of the resultantamplified antibody DNA can indicate the presence of that antibody'scognate antigen on the surface of a cell being examined. Without wishingto be bound by any particular theory, a number of differentphage-displayed blood group antibodies can be contacted simultaneouslyto the same sample of red cells and the differences in antibodynucleotide sequence can be exploited to determine which ones bound andwhich ones did not as demonstrated herein using anti B and anti-Rh(D)antibodies displayed on different phage. Such “multiplexing” is notpossible by agglutination methods as one could never tell whichantibody(ies) caused the agglutination.

That such a methodology is possible, i.e. that the simultaneous bindingof multiple anti-RBC antibodies can be detected by the amplification anddetection of antibody DNA, is demonstrated by the data disclosed hereinwhere a model system comprising phage-displayed anti-blood group B andanti-Rh(D) human monoclonal antibodies was employed. However, theskilled artisan, based upon the disclosure provided herein, wouldreadily appreciate that such “multiplexing” strategy is not limited toany particular antibodies, but can be used to detect multiple red bloodcell antigens using a wide plethora of antibody-displaying phage, whereeach phage comprises a DNA sequence that can be detectably distinguishedfrom the nucleic acid of other phage encoding antibodies havingdifferent specificities, or even phage encoding antibodies having thesame specificities, so long as the nucleic acids of the phage can bedistinguished from one another. Using PCR and agarose gelelectrophoresis to amplify and then detect unique coding sequenceswithin each type of phage particle based on, e.g., size of theamplicons, the data disclosed herein demonstrate that a sample of RBCswas simultaneously phenotyped for B and Rh(D) with extraordinarysensitivity. That is, the single assay detected the equivalent of 20attograms of conventional IgG and required 10,000-fold fewer RBCs (135picoL or about 1500 total RBCs) than a conventional agglutinationreaction.

In practice, however, a rapid, scaleable, and automatable DNA readoutcan be used instead of agarose gel electrophoresis. Many methods arewell-known in the art, and several such methods are discussed more fullyelsewhere herein. Nonetheless, the skilled artisan would understand,once armed with the teachings of the invention, that a wide plethora ofmethods to detect nucleic acids can be used in the methods of theinvention, and the invention is not in any way limited to the methodsexemplified and discussed herein.

Amplification of Phase DNA Using Transcription-Mediated Amplification

In addition to using PCR for phage DNA amplification step (step B inFIG. 1), methods based on detection of transcription of phage antibodyDNA, instead of its amplification, can be used in the methods of theinvention. More specifically, immunodetection by this method has beenused to detect the binding of antibodies to which oligonucleotidescontaining the T7 RNA polymerase promoter site have beenchemically-conjugated with glutaraldehyde as described in Zhang et al.(2001, Proc. Natl. Acad. Sci. USA 98:5497-5502). This technique for thetranscription of DNA that is attached in vivo to an antibody by virtueof its physical association in phage particles can be used as analternative to PCR and other amplification techniques. This technologyhas been termed IDAT, which stands for immuno-detection amplified by T7RNA (Zhang et al., 2001, Proc. Natl. Acad. Sci. USA 98:5497-5502). Byplacing the T7 RNA polymerase promoter site upstream from an arbitrarysequence tag in the phagemid DNA, the addition of T7 RNA polymerase andNTPs rapidly (100 bases per second) produces tag transcripts through theconsecutive and progressive binding of T7 enzymes to their promoter.

Since T7 RNA polymerase binding to RNA products does not occur,amplification is linear not exponential as in PCR. For RBC phenotyping,such linear amplification provides an advantage over PCR (and certainlyover conventional agglutination methods) in that quantitativeinformation (i.e., relative antigen copy number per cell) about multipleantigens can be determined simultaneously from a single sample of cells.An example, among others, of where such quantification can be useful inblood banking is the detection of “weak Rh(D)” phenotypes as reviewed inMollison et al. (1997, In: Blood Transfusion in Clinical Medicine, 10thed., Blackwell Scientific Publications, Oxford, England).

An additional advantage of transcription-based detection methods, suchas, but not limited to, IDAT, over PCR is elimination of temperaturecycling once the antibody phage DNA is released from the particles.Elimination of temperature cycling reactions simplify instrument designand lowers cost of the assay. Nevertheless, PCR and transcriptionmethods each have advantages and disadvantages that are well-known inthe art such that the skilled artisan can readily determine whichmethod, or any other method, can be used for any particular assay andthe conditions desired therefor. This is because PCR, transcription, andmany other methods to detect a nucleic acid, can be used successfully inthe methods of the present invention and the skilled artisan wouldappreciate what method to employ based on art-recognized factors.

Detection of Phase DNA Using Molecular Beacons:

Molecular beacons are stem-and-loop-structured oligonucleotides with afluorescent label at the 5′ end and a universal quencher at the 3′ end(see, e.g., Tyagi and Kramer, 1996, Nature Biotech. 14:303-308; Broude,2002, Trends in Biotechnology 20:249-256). When the stem is closed (inthe absence of complementary nucleic acid), the fluorophore and quencherare in close proximity and fluorescent energy is absorbed by thequencher and fluorescence is quenched and not detectable. In thepresence of complementary nucleic acid, the loop of the beaconhybridizes and the fluorophore and quencher separate such that quenchingdoes not occur. Photons are then emitted from the fluorophore,unquenched, at the wavelength specific for that fluorophore andfluorescence is then detectable. By combining a number of beacons in onetube, each with a different fluorophore at their 5′ ends, multiple DNA(Tyagi et al, 1998, Nature Biotech. 16:49-53) or RNA (de Baar et al.,2001, J. Clin. Microbiol. 39:1895-1902) targets can be simultaneouslydetected by measuring the spectrum of colors emitted from the reactionvessel.

Molecular beacons of two different colors are incorporated into the PCRand transcription reactions to detect the presence of antibody-specificDNA. As described elsewhere herein, anti-Rh(D) and anti-B phage DNA aremodified to contain short DNA sequences that can be amplified (ortranscribed) and subsequently detected using molecular beacons asdescribed elsewhere herein. The design an production of such “beacon”sequences, and nucleic acid sequences comprising sequences“complementary” thereto are well known in the art. Indeed, softwareprograms are commercially available to assist in the design of suchsequences, including the molecular beacon probe sequences complementaryto a sequence of interest.

Further, such beacons and sequences that bind therewith, such as thoseexemplified in FIG. 4, comprise the following sequences: the sequence ofthe “B 140” insert is5′-TGCTATGTCACTTCCCCTTGGTTCTCTCATCTGGCCTGGTGCAATAGGCCCTGCATGCACTGGATGCACTCTATCCCATTCTGCAGCTTCCTCATTGATGGTCTCTTTTAACATTTGCATGGCTGCTTGATGTCCCCCCACT-3′ (SEQ ID NO:3) and the sequence ofthe “D140” insert is5′-TGCTATGTCACTTCCCCTTGGTTCTCTCATCTGGCCTGGTGCAATAGGCCCTGCATGCACTGGATGCACTCTGTTTTACCTCATTATCCTTCTGCCAGCGCTAGCTTTTAACATTTGCATGGCTGCTTGATGTCCCCCCACT-3′ (SEQ ID NO:4). The forward PCRprimer (“PCR-F”) is: 5′-TGCTATGTCACTTCCCCTTGGTTCTCT-3′ (SEQ ID NO:5) andthe reverse PCR primer (“PCR-R”) sequence is:5-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3′ (SEQ ID NO:6). The B-Beacon andD-Beacon sequences are as follows, showing the fluorescent derivativesat the ends and the stem structures in lower case letters. The“B-Beacon” sequence is as follows:6-FAM-gcgagcATCCCATTCTGCAGCTTCCTCATTGATGGTCTCgctcgc-DABCYL (SEQ ID NO:7.The “D-Beacon” is:TAMRA-cgagcGTTTTACCTCATTATCCTTCTGCCAGCGCTAGCgctcgc-DABCYL (SEQ ID NO:8).The upper case letters in the beacon sequences represent the respectivesequences in B140 and D140 to which the beacons anneal. Therefore, theupper case letters are the sequences of the oligonucleotides that areused for the DNA array detection. That is, a

(SEQ ID NO: 9) “B-oligo” is: 5′-ATCCCATTCTGCAGCTTCCTCATTGATGGTCTC-3′,and a (SEQ ID NO: 10) “D-oligo” is:5′-GTTTTACCTCATTATCCTTCTGCCAGCGCTAGC-3′.

The present invention is not limited to these exemplary sequences;rather, the invention encompasses such additional sequences as can bereadily designed by the skilled artisan once armed with the disclosureprovided herein. That is, the design and use of beacon sequences arewell-known in the art and are not discussed further herein and thesequences disclosed herein are merely an example of the sequences thatcan be used to practice the invention. For instance, manyfluorescer-quencher pairs are known in the art, including, but notlimited to, those exemplified herein which encompass6-carboxyfluorescein (6-FAM), 6-carboxytetramethylrhodamine (TAMRA), andDABCYL (a non-fluorescent chromophore that serves as a universalquencher for any fluorophore in a molecular beacon:4-(4-dimethylaminophenylazo)-benzoic acid). Such molecules are wellknown in the art, and are described in, e.g., U.S. Pat. Nos. 6,395,517,and 6,615,063, and are not discussed further herein.

Detection of Phage DNA Using Oligonucleotide Microarrays:

In addition to molecular beacons, hybridization of fluorescent RBC phageantibody amplicons (from PCR) or transcripts (produced using IDAT) toarrays of complementary oligonucleotide probes can be used to indirectlyquantify the amount (if any) of bound antibody in a sample. Further,although the use of conventional methods for hybridization to suchmicroarrays are diffusion limited and may require several hours toobtain adequate fluorescent signals, this process can be accelerated by2-3 orders of magnitude through the application of an electric fieldacross the surface of an inexpensive indium tin oxide-coated glass slideas described in Su et al. (2002, Electrophoresis 23:1551-1557). Thisprocess, known in the art as “electric field-accelerated hybridizationto oligonucleotide microarrays” provides rapid results, e.g., time fromapplication of DNA (or RNA) to readout is less than about 10 minutes.Therefore, electric field-accelerated hybridization can be used tofurther enhance the rapid detection of antigens of interest present on acell (e.g., a red blood cell, a platelet, and the like).

The present invention is not limited to blood typing, but has widepotential uses in many other areas of transfusion medicine, such as, butnot limited to, platelet antigen testing, and has broad application intransplantation immunology (HLA antigen typing) and particularlyforensic medicine, where multiplexing of reactions can provide the mostamount of information from minute amounts of testing samples. Inaddition, the construction of antiglobulin reagents (e.g., anti-IgG,-IgM, -C3 complement component) expressed on phage particles can be usedto perform serum screening for pre-formed anti-RBC antibodies, reversegroup typing, or to perform direct/indirect Coombs tests using amethodology that detects the antiglobulin reagents' associated DNA. Theantiglobulin phage reagents can be isolated from immune murine phagedisplay libraries, or through the cloning of pre-existing hybridomaimmunoglobulin mRNA using techniques well-known in the art.

Detection of Phage DNA Using a Melting Curve Profile

In addition to the other methods for the detection of phage DNA, thedetection of specific nucleic acid sequences encoding a particular phageantibody is accomplished by carrying out a PCR reaction with a set ofprimers that anneals to common sites on the DNA of all phage antibodies,regardless of what antibody DNA is present elsewhere in the DNA. Thedifferences among the phage DNAs encoding the various phage antibodiesare that the length and/or composition of DNA between the primer sitesdiffer between the different phage DNAs. In one aspect, the differencein length is 20 to 50 bases. Therefore, according to the presentinvention, PCR carried out on the DNA from cell-bound phage particleswill generate amplification products of one or more lengths withdiffering base compositions. PCR is conducted in the presence of a dye,such as SYBR Green I, that generates light when bound to double-strandedDNA (such as the PCR products). Thus, the cycles of PCR can be trackedwith respect to the performance of a melting curve using, for example acapillary PCR fluorescent device such as the Roche LIGHTCYCLER(Indianapolis, Ind.). The temperature of the PCR fluorescent device isinitially reduced to around 60° C., then raised in 0.1 degree incrementsup to a temperature of approximately 90° C., measuring the decrease inSYBR Green I green fluorescence as the various lengths of PCRproduct(s)melt. Each PCR product melts at a characteristic temperature,so that a melting curve profile can be examined to determine whichamplicons were present in the PCR reaction, and thus, which phage werebound to the cell, in order to determine which antigens are present on aparticular cell.

Each PCR cycle using the capillary PCR flurorescent device takes a totalof approximately 25 seconds. Therefore, with a modest amount of phageDNA template, only approximately 20 cycles are necessary to generateenough amplicon to obtain a melting temperature. For example, themelting temperature for the PCR product of one phage DNA, anti-B, hasbeen determined using the common set of primers described herein.

Anti-Blood Group B and Anti-Rh(D) Typing Using Phage DNA Analysis

The data disclosed herein demonstrate detection of anti-blood group Band anti-Rh(D) antigens on RBCs using the novel methods of theinvention. That is, two phage displayed human monoclonal antibodies—ananti-blood group B and an anti-Rh(D)—both previously isolated from thepanning of phage display libraries constructed from immunizedindividuals (Chang and Siegel, 2001, Transfusion. 41:6-12; Siegel etal., 1997, J. Immunol. Meth. 206:73-85) were used demonstrating themultiplexing detection of these two antigens.

For the purposes of this study, one antibody (the anti-B termed FB5.7)was expressed as a phage displayed Fab fragment and the other (theanti-Rh(D), termed E1M2) as a single-chain Fv (scFv) fragment (FIG. 2).These antibodies were described previously in Chang and Siegel, 2001,Transfusion. 41:6-12; Siegel et al., 1997, J. Immunol. Meth. 206:73-85;Chang and Siegel, 1998, Blood 91:3066-3078; and U.S. Pat. Nos.5,876,925, 5,985,543, and 6,255,455, all to Siegel. These datademonstrate that various antibody forms (e.g., Fab, scFv, and the like)can be readily used in the methods of the invention.

PCR amplification of the antibody coding regions of the correspondingphagemid DNA was predicted to produce products of different lengths(i.e., 1600 bp and 1000 bp) and agarose gel electrophoresis was then beused to genetically determine the presence of anti-B and/or anti-Rh(D)antibodies instead of conventional antibody-based detection methodsbased on the different sizes of the predicted amplicons.

Before performing binding assays of the phage displayed reagents withRBCs, a series of PCR reactions with serial dilutions of the anti-B oranti-Rh(D) phage preparations were performed to validate the novelgenetic detection method and to determine its sensitivity. PCR of thephagemid antibody coding regions produced the predicted product sizes of1600 bp for the anti-B-encoding Fab DNA and 1000 bp for theanti-Rh(D)-encoding scFv DNA. Remarkably, the sensitivity of detectionwhen visualizing only 10% of the total PCR reaction products, was about100 phage antibody particles. This value represents the equivalent ofonly 1.7×10⁻²² moles or approximately 2×10⁻¹⁷ g of IgG (about 20attograms), a startling level of sensitivity not reached by previousmethods for blood typing.

For PCR amplification of the inserts, the forward primer (“5-prime LC”)was as follows: 5′-AAGACAGCTATCGCGATTG-3′ (SEQ ID NO:1); and the reverseprimer (“GBACK”) was as follows: 5′-GCCCCCTTATTAGCGTTTGCCATC-3′ (SEQ IDNO:2).

To determine whether this genetic assay of using anti-B and anti-Rh(D)phage-displayed antibodies could be used to correctly phenotype RBCs, anexperiment was performed, which demonstrated perfect concordance betweenthe known phenotypes of the reagent RBCs, the conventionalagglutination-based test results performed using the phage antibodies,and the novel genetic testing method results (FIG. 3). Therefore, thedata disclosed herein demonstrate the effectiveness of the novel“phenotyping-by-reagent genotyping” as well as the ability to multiplexphenotype determinations.

Furthermore, using the PCR protocol disclosed herein, the assay isremarkably sensitive given that the results shown in the lanes of theagarose gel depicted in FIG. 3 represent only 10% of the total reactionproduct, and the number of RBCs added to each PCR reaction was onlyabout 1500, or the equivalent of 135 picoL of RBCs. In contrast,conventional methods utilize approximately 10,000 times more RBCs peragglutination assay.

Development of Phage-Displayed Anti-RBC Typing Reagents

The methods utilized to clone, produce, and validate the performancecharacteristics of phage-displayed anti-RBC monoclonal antibodies havebeen the focus of numerous publications (e.g., Siegel, 2002, In: Methodsin Molecular Biology: Antibody Phage Display: Methods and Protocols,vol. 178, pp. 219-226, Aitkem & O'Brien, eds., Humana Press, Totowa,N.J.; Siegel, 2000, In: Phage Display of Proteins and Peptides: ALaboratory Manual, vol. 23, pp. 23.21-23.32, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.; Siegel and Chang, 1997, In: AntibodyEngineering: New Technologies, Applications, & Commercialization, IBC,Boston, Mass.), as well as several issued U.S. patents (see, supra).Specimens (residual peripheral blood, spleen tissue, bone marrow, andthe like) from which RBC antigen specificities other than B, Rh(D), M,and N were isolated, have already been archived using residualdiagnostic patient material.

Rapid and Scaleable Phage Antibody Detection Methodology

The phagemid DNA of anti-RBC blood group, e.g., anti-B and anti-Rh(D),antibodies are modified such that the phage antibodies each contain aunique tag that can be amplified by PCR or transcribed by T7 RNApolymerase and subsequently detected by a corresponding pair of uniquemolecular beacons or microarrayed oligonucleotides. The tags areinserted in the phagemid DNA outside of the anti-B or anti-Rh(D) codingregion so as not to disrupt antibody expression and display on the phagecoat (see, e.g., FIG. 4). A selected number of nucleic acidamplification/detection schemes are performed using the modified set ofanti-RBC phage-displayed antibodies in order to assess the performancecharacteristics in order to maximize the efficiency of rapid,multiplexed, RBC phenotyping.

For the modification of phagemid DNA, B140 and D140 were sequenced.PCR-F and PCR-R, along with B-Beacon/Oligo in kinetic PCR (molecularbeacon) assays to measure the level of HIV gag cDNA (O'Doherty et al.,2000, J. Virol. 74:10074-10080) have been performed. B140 and D140 areligated into anti-B or anti-Rh(D) phagemids using standard cloningtechniques. Antibody-expressing phage particles are produced from theirmodified DNAs and their binding properties are validated as describedpreviously (Chang and Siegel, 1998, Blood 91:3066-3078; Siegel, 2002,In: Methods in Molecular Biology: Antibody Phage Display: Methods andProtocols, vol. 178, pp. 219-226, Aitkem & O'Brien, eds., Humana Press,Totowa, N.J.; Siegel, 2000, In: Phage Display of Proteins and Peptides:A Laboratory Manual, vol. 23, pp. 23.21-23.32, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.; Siegel and Chang, 1997, In: AntibodyEngineering: New Technologies, Applications, & Commercialization, IBC,Boston, Mass.).

Amplification of phage DNA by PCR experiments are performed onRBC/phage-incubated samples as described previously elsewhere herein,except for use of ABI 7700 spectrofluorimetric thermal cycler, additionof one or both of B-BEACON or D-BEACON, or use of PCR fluoresceinlabeling mix (dNTPs spiked with fluorescein dUTP) depending on detectionmethod. For amplification of phage DNA by transcription a series ofexperiments analogous to those performed using PCR are performed usingthe following RNA amplification procedure: Phage particles are heated to94° C. for 2 minutes to denature the phage coat and release thesingle-stranded phagemid DNA. Since T7 RNA polymerase requiresdouble-stranded DNA as template, DNA polymerase I, dNTPs, and the NotI-containing reverse primer used for cloning D140/B140 are used tosynthesize second-strand DNA during RNA synthesis. To initiate RNAamplification, amplification buffer containing T7 RNA polymerase (Zhanget al., 2001, Proc. Natl. Acad. Sci. USA 98:5497-5502) is added in thepresence of one or both molecular beacons or fluorescein-12-UTPdepending on the detection method as described below.

For the detection of amplified phage DNA using molecular beacons,B-Beacon (FAM-labeled) and D-Beacon (TAMRA-labeled) stem-and-loopstructures are present both singly and in combination during PCRamplicon formation and during RNA transcription. The shortesttime-to-positivity (fewest PCR cycles/shortest time for RNAtranscription), where positivity is at least 2 logs of fluorescenceabove background, is determined. Initially, sensitivity assays areperformed using serial dilutions of phage and followed by bindingexperiments using antigen-negative and positive RBCs. The ability tomultiplex reactions with anti-B and anti-Rh(D) is assessed and titeringnumerous variables are examined including relative concentrations ofeach beacon, amount of inputted phage antibodies, number of RBCs, timeof RBC/phage incubation, and number of washes. In addition, the effecton the fluorescent signal as a function of antigen copy number per RBCis assessed (e.g., compare Rh(D) phenotypes R₂R₂, R₁R₁, R₁r, D^(w),partial Rh(D), and the like.) (Mollison et al., 1997, In: BloodTransfusion in Clinical Medicine, 10th ed., Blackwell ScientificPublications, Oxford, England) and quantification of antigen densitywith exponential (PCR) and linear (transcription) amplification arecompared.

For the detection of amplified phage DNA on electric-field enhancedoligonucleotide microarrays, oligonucleotides corresponding to thehybridizing nucleotides of B-Beacon and D-Beacon are synthesized andapplied to indium tin oxide-coated glass slides using an arrayer. Slidesare processed, incubated with fluorescently-labeled PCR amplicons or RNAtranscripts, and washed as described (Su et al., 2002, Electrophoresis23:1551-1557) and analyzed using a ScanArray 5000 microarray scanner.

Similar to the approach taken in the molecular beacon experimentsdescribed above, the detection of anti-B- and -Rh(D)-associated phageDNA in the shortest time is optimized by varying similar parameters.Because RBCs with and without each antigen are included, test sampleswith one or both (or neither) phage antibody, and a microarray withmultiple spots, a number of internal positive and negative controls arepresent that will permit an accurate assessment of signal/noise ratio.Based on previous experience, it is estimated that less than 10 minutesfrom the time of sample application to hybridization and readout arerequired.

Routine molecular cloning methods are used and troubleshooting isstraightforward. Furthermore, it is unlikely that there is any adverseaffect on antibody expression or display resulting from the introductionof B140/D140. Other nucleotide sequences have been successfully clonedinto the Not I site of pComb3X without any untoward effects.Furthermore, there are other convenient unique restriction sites intowhich B140/D140 (or an alternative set of tags) can be cloned, ifnecessary or desired. The important features of the assays is therelative time-to-positivity for the amplification/detection strategyused and it lends itself to multiplexing of RBC phenotyping. PCR studiesdisclosed herein demonstrated sensitivity and specificity. Thetranscription procedure, although linear, offers the simplicity ofisothermal amplification reactions and, with an input of 10⁷-10⁹template DNAs per sample, sensitivity will likely not be a limitingfactor. Indeed, previous studies utilizing transcription methods withglutaraldehyde-conjugated oligonucleotide/monoclonal antibodiesdemonstrated 10⁹ to 10¹¹-fold greater sensitivity than ELISA assays andenhanced chemiluminescence-Western blot assays, respectively, with areported ability to detect as little as a few copies of antigen in acell lysate (Zhang et al., 2001, Proc. Natl. Acad. Sci. USA98:5497-5502).

Further, the methods disclosed herein present vast improvement overinstruments, such as, but not limited to, the Olympus™ PK7200 automatedanalyzer, which are considered state-of-the-art for a device that useshemagglutination technology. This is because with a reported throughputof several hundred specimens per hour, the methods disclosed hereinrepresent feasible and ultimately superior in that time-to-positivity(including RBC/phage incubation times) is in the 30-minute range,reactions take place in a 96-well format, and multiple antigendeterminations (multiplexing) can take place in a single well.

Detection of Auto- and Alloantibodies in Serum

This assay is performed in a manner similar to the standard indirectantiglobulin test (see, e.g., Mollison, 1997, In: Blood Transfusion inClinical Medicine, 10th ed., Blackwell Scientific Publications, Oxford,England) with the substitution of antiglobulin expressing phageparticles for the conventional antiglobulin reagent, and the detectionof bound phage reagent as disclosed herein based on detection of a knownnucleic acid sequence present in the bacteriophage. Briefly, members ofa panel of reagent red blood cells of known antigen composition are eachincubated with an aliquot of patient sera. Cells are washed to removenon-specifically bound antibodies and the cells are then contacted withbacteriophage displaying an antiglobulin reagent. The antiglobulinreagent can be specific for all human immunoglobulin isotypes ifdesired, or specific for only one class such as IgM or IgG. Usingalgorithms well known in the field of immunohematology, the specificityor specificities of anti-red blood cell antibodies present in thepatient sera is determined based on the pattern of reactivity of serawith panel red blood cells.

Detection of Antibody or Complement Fragments on Red Blood Cells

This assay is performed in a manner similar to the standard directantiglobulin test (see, e.g., Mollison, 1997, Blood Transfusion inClinical Medicine, 10th ed., Blackwell Scientific Publications, Oxford,England) with the substitution of antiglobulin expressing phageparticles for the conventional antiglobulin reagent, and the detectionof bound phage reagent as disclosed herein based on detection of a knownnucleic acid sequence present in the bacteriophage. Briefly, a sample ofred blood cells is washed to remove non-specifically bound antibodiesand then contacted with bacteriophage displaying an antiglobulin reagenton its surface. The antiglobulin reagent preparation can comprisemolecules specific for IgG, for complement C3d, or both (e.g., anti-IgGantibody, anti-C3d antibody, both, and the like). Where there is humanantibody or complement bound with the cell, the bacteriophage binds viathe antiglobulin reagent displayed by the phage. The presence of phagespecifically bound with the cell (via binding with the human antibody orcomplement on the cell) is then detected as disclosed herein based ondetection of a known nucleic acid sequence present in the bacteriophage.

Performing Donor/Recipient Compatibility Testing

This assay is performed in a manner similar to the standard Coombscrossmatch test (see, e.g., Mollison, 1997, In: Blood Transfusion inClinical Medicine, 10th ed., Blackwell Scientific Publications, Oxford,England) with the substitution of antiglobulin expressing phageparticles for the conventional antiglobulin reagent, and the detectionof bound phage reagent as disclosed herein based on detection of a knownnucleic acid sequence present in the bacteriophage. Briefly, the methodcomprises contacting a sample of donor red blood cells with a patientserum sample. The cells are washed to remove non-specifically boundantibodies and the cell is then contacted with bacteriophage displayingan antiglobulin reagent (e.g., anti-IgM or anti-IgG) on its surface.Where there is human antibody bound with the cell, such as would be thecase with an incompatible crossmatch, the bacteriophage binds via theantiglobulin reagent displayed by the phage. The presence of phagespecifically bound with the cell (via binding with the human antibody onthe cell) is detected as disclosed herein based on detection of a knownnucleic acid sequence present in the bacteriophage.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed:
 1. A method of detecting the presence of a plurality of known antigen-bearing moieties on a cell in a single tube assay, said method comprising the steps of: 1) antigen detection, 2) amplification of phage nucleic acid, and 3) detection of phage nucleic acid, wherein said antigen detection step consists of: contacting a cell with a plurality of bacteriophage types, each expressing an antibody known to specifically bind with an antigen-bearing moiety wherein each of said bacteriophage type comprises a nucleic acid comprising a nucleic acid sequence that encodes the antibody, wherein the sequence that encodes the antibody is known and at least a portion of the sequence is correlated to the phenotype of one of said plurality of known antigen-bearing moieties, wherein the sequence that encodes the antibody from each of said bacteriophage type is detectably different from the sequence that encodes the antibody from all other bacteriophage types; wherein said amplification step comprises conducting PCR reactions using nucleic acid primers that anneal to said nucleic acids in each of said bacteriophage types to generate amplified products, wherein the amplified products comprise at least a portion of the sequence that encodes the antibody; and wherein said detection of phage nucleic acid step consists of detecting the binding of each of said bacteriophage type with each of said antigen-bearing moiety by detecting the presence of each of said amplified product using a melting curve profile, wherein detecting each of said amplified product detects the presence and phenotype of each of said antigen-bearing moiety on said cell.
 2. The method of claim 1, said method further comprising washing said cell between said antigen detection step and said amplification step.
 3. The method of claim 1, wherein the melting curve profile is determined using a capillary PCR fluorescent device.
 4. The method of claim 1, wherein said cell is a red blood cell and at least one of said plurality of antigen-bearing moieties is a red blood cell antigen.
 5. The method of claim 4, wherein said red blood cell antigen is selected from the group consisting of A, B, Rh(D), Rh(C), Rh(c), Rh(E), Rh(e), K, Fy^(a), Fy^(b), M, N, S, s, Jk^(a), and Jk^(b).
 6. The method of claim 1, wherein said cell is a white blood cell and at least one of said plurality of antigen-bearing moieties is selected from the group consisting of a lymphocyte antigen, a monocyte antigen, and a granulocyte antigen.
 7. The method of claim 1, wherein said cell is a platelet and further wherein-at least one of said plurality of antigen-bearing moieties is a platelet antigen.
 8. The method of claim 7, wherein said platelet antigen is selected from the group consisting of HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b, HPA-6b, HPA-7b, HPA-8b, HPA-9b, HPA-10b, Gov^(a), and Gov^(b).
 9. A method of detecting the presence of a known antigen-bearing moieties on a cell, said method consisting, a) contacting a cell with a plurality of bacteriophage types, each expressing an antibody known to specifically bind with said specific antigen-bearing moiety wherein said bacteriophage types comprises a nucleic acid comprising a nucleic acid sequence that encodes the antibody and wherein the sequence that encodes the antibody of said nucleic acid is known; b) denaturing any of said bacteriophage types specifically bound with said cell to release said nucleic acid; and c) detecting said released nucleic acids using a melting curve profile, wherein detecting said nucleic acids detects the presence of said antigen-bearing moieties on said cell. 